WO2012047630A2

WO2012047630A2 - N-alkyl or n-aryl substituted guanide and biguanide compounds and methods of their use

Info

Publication number: WO2012047630A2
Application number: PCT/US2011/053401
Authority: WO
Inventors: Martin Teintze; Royce A. Wilkinson; Mohamed Labib
Original assignee: Martin Teintze; Wilkinson Royce A; Mohamed Labib
Priority date: 2010-09-27
Filing date: 2011-09-27
Publication date: 2012-04-12
Also published as: WO2012047630A3

Abstract

The present invention provides N-aryl or N-alkyl guanide and biguanide containing compounds having the structure X. or the structure XI. These compounds may be synthesized by the addition of the respective guanide, biguanide or phenylguanide groups to polyamines containing primary and/or secondary amines. The present invention includes methods of using these compounds for the treatment and/or prevention of bacterial infection, HIV infection, and cancer and for the promotion of stem cell mobilization, wound healing, hematopoiesis, and skin rejuvenation.

Description

N-Alkyl or N-Aryl Substituted Guanide and Biguanide Compounds and

Methods of Their Use

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application Serial No.

61/386,855, filed September 27, 2010, which is herein incorporated by reference in its entirety.

GOVERNMENT RIGHTS STATEMENT

This invention was made with government support under grants AI076965,

AI064107, and P20 R 16455 from the National Institutes of Health. The government has certain rights in the invention.

TECHNICAL FIELD

This application relates to compositions, methods to prevent, inhibit and/or treat bacterial infection, HIV infection, and cancer, and methods to promote stem cell

mobilization, wound healing, hematopoiesis, and skin rejuvenation.

BACKGROUND

Viruses and bacteria are always evolving to develop resistance to drugs that are in clinical use, so new lead compounds with novel mechanisms of action are needed. In addition, HIV and cancer therapies all have undesired side-effects. There is demand for new compounds with low cytotoxicity and relatively few side-effects to more effectively treat and prevent these life-threatening diseases.

Bacteria are particularly adept at developing mechanisms of resistance to antibiotics, and opportunistic pathogens with innate resistance to antibiotics have been emerging. In developed countries such as the United States, major bacteria that have acquired multiple drug resistance include the ESKAPE pathogens, aptly named for their ability to escape the effects of our current antimicrobial drugs. These include Enterococcus faecium,

Staphylococcus aureus (S. aureus), Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter species (Boucher, et al. (2009) Clin Infect Dis. 48: 1-12). An estimated 12 million outpatient visits occur annually in the US because of S. aureus skin and soft-tissue infections (McCaiget al. (2006) Emerg Infect Dis. 12: 1715-23), and invasive methicillin-resistant S. aureus (MRSA) disease was responsible for nearly 19,000 deaths during 2005 (Klevenset al. (2007) JAMA. 298: 1763-71). MRSA was once thought to be problematic only in healthcare settings, but are now commonly contracted by otherwise healthy individuals with no prior exposure to healthcare settings. MRSA are resistant to the entire class of beta-lactam antibiotics including penicillins, cephalosporins, and monobactams. MRSA strains are also becoming resistant to additional classes of antibiotics, including macrolides (such as erythromycin and clindamycin) and quinolones (such as levofloxacin) (Lowy (2003) J Clin Invest. I l l : 1265-73), as well as the glycopeptide vancomycin, the common drug of last resort (MMWR Morb Mortal Wkly Rep. (2002) 51 :902). Linezolid, quinupristin/dalfopristin (synercid), daptomycin, and tigecycline have been used to treat infections that do not respond to vancomycin, but the development of resistance to these drugs is also likely. Given how quickly drug-resistance spreads in S. aureus and other bacterial species, there is a growing need to develop new treatment options for bacterial infections.

Moreover, new therapeutics with novel mechanisms of action are needed for treatment of human immunodeficiency virus type 1 (HIV-1) infections. Maraviroc, a small molecule antagonist of the CCR5 receptor was the first drug of its type to be approved, and it has proven to be effective against "R5" HIV that uses the CCR5 co-receptor (Dorr et al.

(2005) Antimicrob Agents Chemother 49:4721-32). However, most patients that have failed conventional therapies also harbor "X4" HIV that uses CXCR4 or dual-tropic viruses that can use either CCR5 or CXCR4. The appearance of the X4 viruses is associated with progression to AIDS symptoms (Connor et al. (1997) J Exp Med 185:621-8), and drug resistance is more often linked to CXCR4-utilizing than to CCR5 -utilizing HIV (Wagner et al. (2008) Aids

22:2393-5). Therefore, there is also a need to develop effective drugs that block CXCR4. So far, none of the CXCR4 antagonists in development have shown promise in HIV clinical trials.

In addition, there is recognition of the importance of chemokines and their role in biology. Agents and methods that functions as antagonists or agonists that interfere with the binding of chemokines with their natural ligands have shown utility in stem cell mobilization and show promise as new therapies for purposes including wound healing, hematopoiesis, and skin rejuvenation. SUMMARY OF THE INVENTION

The present invention includes a compound having the following structure:

or a pharmaceutically-acceptable salt or ester thereof, wherein:

Ri is selected from the group consisting of:

hydrogen and

R₂ is selected from the group consisting of:

and

wherein each instance of D is independently selected from the group consisting of: hydro linear or branched Ci-C₆ alkyl, linear or branched C₂-C₆ alkenyl, linear or branched C₂-C alkynyl, and

wherein said linear or branched alkyl, alkenyl, or alkynyls may be optionally substituted with one or more -OH; and each of which may include one or more spacer moieties selected from the group consisting of O, S, NH(C=0), (C=0)NH, 0(C=0), (C=0)0 or (C=0); wherein each instance of K is independently selected from the group consisting of: hydrogen, linear or branched Ci-C₆ alkyl, linear or branched C₂-C₆ alkenyl, linear or branched C₂-C₆ alkynyl group and

wherein said linear or branched alkyl, alkenyl or alkynyls may include one or more spacer moieties selected from the group consisting of O, S, NH(C=0), (C=0)NH, 0(C=0), (C=0)0 or (C=0); each instance of A is independently selected from the group consisting of: linear or branched C₁-C₁₂ alkyl, linear or branched C₂-C₁₂ alkenyl, or linear or branched C₂-C₁₂ alkynyl, wherein said linear or branched alkyl, alkenyl or alkynyls may include one or more spacer moieties selected from the group consisting of: O, S, NH(C=0), (C=0)NH, 0(C=0), (C=0)0 or (C=0); each instance of R is independently selected from the group consisting of: hydrogen, Ci-C₆ alkyl, -NH₂,

wherein M is selected from the group consisting of phenyl, napthalene, and a monocyclic or bicyclic heteroaromatic group,

wherein phenyl, napthalene, and the monocyclic or bicyclic heteroaromatic group are each optionally substituted with one or more substituents independently selected from the group consisting of halo, hydroxy, alkyl, linear or branched Ci-C₆ alkyl, linear or branched C₂-C₆ alkenyl, linear or branched C₂-C₆ alkynyl, cycloalkyl, haloalkyl, hydroxyalkyl, alkoxy and alkoxyalkyl;

with the proviso that at least one R must be one of structures I-III.

In another embodiment, the present invention is a compound having the following structure:

or a pharmaceutically-acceptable salt or ester thereof, wherein:

Z is selected from the group consisting of straight chained or branched C₁-C₁₂ alkyl, straight chained or branched C₂-C₁₂ alkenyl, straight chained or branched C₂-C₁₂ alkynyl; wherein said straight chained or branched alkyl, alkenyl or alkynyls may include one or more spacer moieties selected from the group consisting of O, S, NH(C=0), (C=0)NH, 0(C=0), (C=0)0, (C=0) and

wherein each instance of Y is independently selected from the group consisting of hydrogen,

IA.

wherein phenyl, napthalene, and the monocyclic or bicyclic heteroaromatic group are each optionally substituted with one or more substituents independently selected from the group consisting of halo, hydroxy, linear or branched Ci-C₆ alkyl, linear or branched C₂-C6 alkenyl, linear or branched C₂-C₆ alkynyl, cycloalkyl, haloalkyl, hydroxyalkyl, alkoxy and

alkoxyalkyl; and each instance of R is independently selected from the group consisting of: hydrogen, -CH₃, -NH₂,

II.

III.

wherein M

is selected from the group consisting of phenyl, napthalene, and a monocyclic or bicyclic heteroaromatic group,

alkoxyalkyl and

with the proviso that when Z does not include at least one spacer moiety that is

wherein Y is independently selected from the group consisting of structures IA-IIIA, at least one R must be one of structures I-III.

In another embodiment, the invention is a composition comprising:

a compound having the following structure:

or a pharmaceutically-acceptable salt or ester thereof, wherein: Ri is selected from the group consisting of: hydrogen and

R₂ is selected from the group consisting of:

and

wherein phenyl, napthalene, and the monocyclic or bicyclic heteroaromatic group are each optionally substituted with one or more substituents independently selected from the group consisting of halo, hydroxy, linear or branched Ci-C₆ alkyl, linear or branched C₂-C₆ alkenyl, linear or branched C₂-C₆ alkynyl, cycloalkyl, haloalkyl, hydroxyalkyl, alkoxy and

alkoxyalkyl;

and one or more pharmaceutically acceptable excipients.

The invention in another embodiment is a composition comprising a compound having the following structure:

wherein:

IA.

IIA.

IIIA.

wherein phenyl, napthalene, and the monocyclic or bicyclic heteroaromatic group are each optionally substituted with one or more substituents independently selected from the group consisting of halo, hydroxy, linear or branched Ci-C₆ alkyl, linear or branched C₂-C₆ alkenyl, linear or branched C₂-C6 alkynyl, cycloalkyl, haloalkyl, hydroxyalkyl, alkoxy and

II.

wherein M

alkoxyalkyl;

and

and one or more pharmaceutically acceptable excipients.

The invention, in another embodiment, comprises a compound that binds to a G- protein coupled receptors such as those that act as co-receptors for HIV-1 infection. In one embodiment, a compound of the present invention binds the G-protein coupled receptor(s) CXCR4 and/or CCR5. In another embodiment, a compound of the present invention binds CXCR4 with an IC₅o less than about 500 nM, about 450 nM, about 400 nM, about 350 nM, about 300 nM, about 250 nM, about 200 nM, about 150 nM, about 100 nM, about 50 nM, about 25 nM about 10 nM, about 5 nM, about 2.5 nM, or about 1 nM. In one embodiment, a compound of the present invention binds CXCR4 with an IC₅₀ less than about 500 μΜ, about 450 μΜ, about 400 μΜ, about 350 μΜ, about 300 μΜ, about 250 μΜ, about 200 μΜ, about 150 μΜ, about 100 μΜ, about 50 μΜ, about 25 μΜ, about 10 μΜ, about 5 μΜ, about 2.5 μΜ or about 1 μΜ in competition cross-link inhibition assays with T-140.

In one embodiment, the compounds of the present invention are used in a method of preventing or treating bacterial infection, an HIV infection, and/or cancer. In another embodiment, the invention is a method of administering an effective amount of a compound of the present invention to a patient in need thereof. The present invention includes a method of administering an effective amount of compound that acts as a cytokine antagonist; to improve skin rejuvenation; to increase stem-cell mobilization; to increase hematopoiesis; to improve wound healing; to treat, inhibit and/or prevent HIV; to treat, inhibit and/or prevent cancer; to treat, inhibit and/or prevent a bacterial infection; and/or to treat, inhibit and/or prevent a viral infection. In one embodiment, the compounds of the present invention may be used to treat a bacterial infection resulting from an infection of Acinetobacter baumannii, Burkholderia Cepacia, Enterococcus faecalis, Escherichia coli, Pseudomonas aeruginosa and/or Staphylococcus aureus. In another embodiment, the compounds of the present invention may be used to treat an infection of methicillin-resistant Staphylococcus aureus (MRS A). In another embodiment, the compounds of the present invention may be used to treat an HIV infection resulting from an infection by the R5 and/or X4 strain(s) of HIV-1.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 depicts the structure of starting material polyamines that may be used to synthesize guanide, biguanide, and phenylguanide derivatives.

Figure 2 depicts an exemplary synthetic reaction scheme for preparation of the compounds of the present invention.

Figure 3 depicts a mechanism of formation of an O-methylisourea reaction byproduct.

Figure 4 depicts byproducts formed during the synthesis of DETA phenylguanide.

Figure 5 depicts bacterial counts in mice treated or untreated with THAM-3G

(Figure 5 A) and with THAM-30G (Figure 5B) and then challenged with MRS A.

Figure 6 depicts representative CXCR4-T140 cross-link inhibition experiment gel (Figure 6A) and graphical analysis (Figure 6B).

Figure 7 depicts an initial anti-HIV activity screen using titered viral supernatants for HIV clones NL4-3 (Figure 7A), 92HT599 (Figure 7B), 92HT594 (Figure 7C), and Ba-L (Figure 7D) to infect TZM-bl cells. Experiments were performed in triplicate with errors shown as ± SEM. RLU = relative luminescence units. Figure 8 depicts HIV inhibition dose-response trials. Spermine (·), spermine phenylguanide (o), spermidine (T), and spermidine phenylguanide (Δ) were tested for dose- response activity in the TZM-bl luciferase anti-HIV assay with HIV clones NL4-3 (Figure 8A), 92HT599 (Figure 8B), MN (Figure 8C), and Ba-L (Figure 8D). Experiments were performed in triplicate with errors shown as ± SEM. RLU = relative luminescence units.

Figure 9 depicts spermidine phenylguanide activity and cytotoxicity. HIV inhibition activity of spermidine phenylguanide (·) and spermidine (o) against three CXCR4 specific HIV clones, NL4-3 (Figure 9A), 92HT599 (Figure 9B), and MN (Figure 9C) using the TZM-bl assay is shown. Experiments were performed in triplicate with errors shown as ± SEM. RLU = relative luminescence units. Figure 9D presents an MTS assay measuring the cytotoxicity (% of control cells) for spermidine phenylguanide (·) and spermidine (o) against the TZM-bl cells. Experiments were performed in duplicate with errors shown as ± SEM.

DETAILED DESCRIPTION

In one embodiment, the present invention is a method of synthesizing guanide compounds, biguanide compounds, or phenylguanide compounds comprising addition of a guanide reagent, a biguanide reagent or a phenylguanide reagent to a reactive primary or secondary amine. The guanide reagent may be O-methylisourea sulfate salt, the biguanide reagent may be S-methyl-guanylisothiouronium iodide, and the phenylguanide reagent may be S-methyl-N-phenylisothiouronium iodide. The resulting poly-guanide, biguanide, or phenylguanide compounds may be linear, branched, cyclic or dendrimeric. In one embodiment, the method of synthesizing guanide compounds, biguanide compounds, or arylguanide compounds (such as phenyl guanide compounds) comprises addition of a guanide reagent, a biguanide reagent or a arylguanide reagent to a reactive primary or secondary amine on a linear or branched polymer.

As used herein, the term "guanide compound" may be used interchangeably with "guanide derivative" and means a chemical compound containing the substituent designated A below. The term "biguanide compound" may be used interchangeably with "biguanide derivative" and means a chemical compound containing the substituent designated B below.

As used herein, the term "aryl" by itself or as part of another group refers to monocyclic or bicyclic aromatic groups containing from 6 to 12 carbons in the ring portion, and may be optionally substituted with one or more substituents. Accordingly, the term "arylguanide compound" may be used interchangeably with "arylguanide derivative" and means a chemical compound containing the subsitiutent designated directly below

wherein M is s selected from the group consisting of phenyl, napthalene, and a monocyclic or bicyclic heteroaromatic group; and wherein phenyl, napthalene, and the monocyclic or bicyclic heteroaromatic group are each optionally substituted with one or more substituents independently selected from the group consisting of halo, hydroxy, alkyl, linear or branched C1-C6 alkyl, linear or branched C2-C6 alkenyl, linear or branched C2-C6 alkynyl, cycloalkyl, haloalkyl, hydroxyalkyl, alkoxy and alkoxyalkyl. An "arylguanide" reagent is a compound that is chemically reactive with a primary or secondary amine yielding the arylguanide substituent subsitutuent above. The term "phenylguanide compound" may thus be used interchangeably with "phenylguanide derivative" and means a chemical compound containing the substituent designated C below.

A "guanide reagent," a "biguanide reagent," and a "phenylguanide reagent" are compounds chemically reactive with a primary or secondary amine yielding substituent A, substituent B, and substituent C, respectively.

Guanide, biguanide, and phenylguanide compounds, for example, can be synthesized by addition to reactive primary or secondary amines as shown in the scheme above and in Figure 2. In this manner, polyamine compounds can be converted to the corresponding poly- guanides, biguanides, or arylguanides. The new method of the present invention allows for linear, branched, or dendrimeric forms to be synthesized in a controlled fashion.

The reagents for addition of the desired groups are either commercially available or can be readily synthesized. O-methylisourea is commercially available, while the S-methyl- N-guanylisourea and S-methyl-N-phenylisourea can be synthesized by the addition of methyl iodide to the commercially available thioureas. Additional alkyl or aryl derivatives may also be produced by the addition of thiocyanate to the corresponding amines to yield intermediate thioureas which can be converted to the S-methylisothioureas by the addition of methyliodide as shown below.

The term "alkyl" herein alone or as part of another group refers to a monovalent alkane (hydrocarbon) derived radical containing from 1 to 12 carbon atoms unless otherwise defined. In some embodiments, the alkyl groups are lower alkyl groups having from 1 to 6 carbon atoms. An alkyl group is an optionally substituted straight, branched or cyclic saturated hydrocarbon group. Alkyl groups may be substituted at any available point of attachment. An alkyl group substituted with another alkyl group is also referred to as a

"branched alkyl group". Exemplary alkyl groups include methyl, ethyl, propyl, isopropyl, n- butyl, t-butyl, isobutyl, pentyl, hexyl, isohexyl, heptyl, 4,4-dimethylpentyl, octyl, 2,2,4- trimethylpentyl, nonyl, decyl, undecyl, dodecyl, and the like. Exemplary substituents include but are not limited to one or more of the following groups: alkyl, cycloalkyl,

heterocycloalkyl,— CN, aryl, heteroaryl, halo (such as F, CI, Br, I), haloalkyl (such as CC1 3 or CF 3 ), hydroxyl, alkoxy, alkylthio, alkylamino,— COOH,— COOR,— C(0)R,— OCOR, amino, carbamoyl (— NHCOOR— or— OCONHR— ), urea (— NHCONHR— ) or thiol (— SH). The term "alkenyl" herein alone or as part of another group refers to a straight, branched or cyclic hydrocarbon radical containing from 2 to 12 carbon atoms and at least one carbon to carbon double bond. Alkenyl groups may also be substituted at any available point of attachment. Exemplary substituents for alkenyl groups include those listed above for alkyl groups.

The term "alkynyl" herein alone or as part of another group refers to a straight, branched or cyclic hydrocarbon radical containing from 2 to 12 carbon atoms and at least one carbon to carbon triple bond. Alkynyl groups may also be substituted at any available point of attachment. Exemplary substituents for alkynyl groups include those listed above for alkyl groups.

The term "alkoxy" or "alkyloxy" refers to any of the above alkyl groups linked to an oxygen atom. Typical examples are methoxy, ethoxy, isopropyloxy, sec-butyloxy, and t- butyloxy.

The term "halogen" or "halo" as employed herein by itself or as part of another group refers to chlorine, bromine, fluorine or iodine.

The term "hydroxyalkyl" as employed herein refers to any of the above alkyl groups wherein one or more hydrogens thereof are substituted by one or more hydroxyl moieties.

The term "haloalkyl" as employed herein refers to any of the above alkyl groups wherein one or more hydrogens thereof are substituted by one or more halo moieties. Typical examples include fluoromethyl, difluoromethyl, trifluoromethyl, trichloroethyl, trifluoroethyl, fluoropropyl, and bromobutyl.

The term "cycloalkyl" as employed herein by itself or as part of another group refers to cycloalkyl groups containing 3 to 9 carbon atoms. Typical examples are cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl and cyclononyl.

The numbers in the subscript after the symbol "C" define the number of carbon atoms a particular group can contain. For example "C _1-6 alkyl" means a straight or branched saturated carbon chain having from one to six carbon atoms; examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, t-butyl, n-pentyl, sec-pentyl, isopentyl, and n-hexyl. Depending on the context, "C i_₆ alkyl" can also refer to C _1-6 alkylene which bridges two groups; examples include propane-l,3-diyl, butane- 1,4-diyl, 2-methyl-butane-l,4-diyl, etc. "C 2-6 alkenyl" means a straight or branched carbon chain having at least one carbon- carbon double bond, and having from two to six carbon atoms; examples include ethenyl, propenyl, isopropenyl, butenyl, isobutenyl, pentenyl, and hexenyl. Depending on the context, "C 2-6 alkenyl" can also refer to C 2-6 alkenediyl which bridges two groups; examples include ethylene- 1,2-diyl (vinylene), 2-methyl-2-butene-l,4-diyl, 2-hexene-l,6-diyl, etc. "C 2-6 alkynyl" means a straight or branched carbon chain having at least one carbon-carbon triple bond, and from two to six carbon atoms; examples include ethynyl, propynyl, butynyl, pentynyl, and hexynyl.

The term "heterocycle" (and variations thereof such as "heterocyclic" or

"heterocyclyl") broadly refers to (i) a stable 4- to 8-membered, saturated or unsaturated monocyclic ring, or (ii) a stable 7- to 12-membered bicyclic ring system, wherein each ring in (ii) is independent of, or fused to, the other ring or rings and each ring is saturated or unsaturated, and the monocyclic ring or bicyclic ring system contains one or more heteroatoms (e.g., from 1 to 6 heteroatoms, or from 1 to 4 heteroatoms) selected from N, O and S and a balance of carbon atoms (the monocyclic ring typically contains at least one carbon atom and the ring systems typically contain at least two carbon atoms); and wherein any one or more of the nitrogen and sulfur heteroatoms is optionally oxidized, and any one or more of the nitrogen heteroatoms is optionally quaternized. Unless otherwise specified, the heterocyclic ring may be attached at any heteroatom or carbon atom, provided that attachment results in the creation of a stable structure. Unless otherwise specified, when the heterocyclic ring has substituents, it is understood that the substituents may be attached to any atom in the ring, whether a heteroatom or a carbon atom. Saturated heterocyclics form a subset of the heterocycles; i.e., the term "saturated heterocyclic" generally refers to a heterocycle as defined above in which the entire ring system (whether mono- or poly-cyclic) is saturated. The term "saturated heterocyclic ring" refers to a 4- to 8-membered saturated monocyclic ring or a stable 7- to 12-membered bicyclic ring system which consists of carbon atoms and one or more heteroatoms selected from N, O and S. Representative examples include piperidinyl, piperazinyl, azepanyl, pyrrolidinyl, pyrazolidinyl, imidazolidinyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiomorpholinyl, thiazolidinyl, isothiazolidinyl, and tetrahydrofuryl (or tetrahydrofuranyl). Heteroaromatics form another subset of the heterocycles; i.e., the term "heteroaromatic" (alternatively "heteroaryl") generally refers to a heterocycle as defined above in which the entire ring system (whether mono- or poly-cyclic) is an aromatic ring system. The term "heteroaromatic ring" or "heteroaromatic group" refers a 5- or 6-membered monocyclic aromatic ring or a 7- to 12-membered bicyclic which consists of carbon atoms and one or more heteroatoms selected from N, O and S. In the case of substituted heteroaryl rings containing at least one nitrogen atom (e.g., pyridine), such substitutions can be those resulting in N-oxide formation. Representative examples of heteroaromatic rings include pyridyl, pyrrolyl, pyrazinyl, pyrimidinyl, pyridazinyl, thienyl (or thiophenyl), thiazolyl, furanyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isooxazolyl, oxadiazolyl, thiazolyl, isothiazolyl, and thiadiazolyl.

Representative examples of bicyclic heterocycles include benzotriazolyl, indolyl, isoindolyl, indazolyl, indolinyl, isoindolinyl, quinoxalinyl, quinazolinyl, cinnolinyl, chromanyl, isochromanyl, tetrahydroquinolinyl, quinolinyl, tetrahydroisoquinolinyl, isoquinolinyl, 2,3-dihydrobenzofuranyl, 2,3-dihydrobenzo-l,4-dioxinyl, imidazo(2,l-b) (l,3)thiazole, and benzo-l,3-dioxolyl.

The term "spacer" or "spacer moiety" is used herein to refer to an atom or a collection of atoms which may interrupt or be interspersed within a moiety or substituent group. The spacer moieties of the invention may be hydro lyrically stable or may include a

physiologically hydro lyzable or enzymatically degradable linkage.

The term "pharmaceutically acceptable salt" as used herein refers to a salt of an acid and a basic nitrogen atom of a compound of the present invention. The term

"pharmaceutically acceptable salt" may also include a hydrate of a compound or its pharmaceutically acceptable salt of the present invention. Exemplary salts include, but are not limited to, sulfate, citrate, acetate, oxalate, chloride, hydrochloride, bromide, hydrobromide, iodide, nitrate, bisulfate, phosphate, acid phosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, gentisinate, gluconate, glucaronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, camphorsulfonate, napthalenesulfonate, propionate, succinate, fumarate, maleate, malonate, mandelate, malate, palmitate, aspartate, phthalate, and pamoate. The term "pharmaceutically acceptable salt" as used herein also refers to a salt of a compound of the present invention having an acidic functional group, such as a carboxylic acid functional group, and a base. Exemplary bases include, but are not limited to, hydroxide of alkali metals including sodium, potassium, and lithium; hydroxides of alkaline earth metals such as calcium and magnesium; hydroxides of other metals, such as aluminum and zinc; ammonia, organic amines such as unsubstituted or hydroxyl-substituted mono-, di-, or tri-alkylamines, dicyclohexylamine; tributyl amine; pyridine; N-methyl, N-ethylamine; diethylamine; triethylamine; mono-, bis-, or tris-(2-OH— (C 1 -C 6 )-alkylamine), such as N,N-dimethyl-N-(2-hydroxyethyl)amine or tri-(2-hydroxy)amine; N-methyl-D-glucamine; morpholine; thiomorpholine; piperidine; pyrrolidine; and amino acids such as arginine, lysine, and the like.

The term "solvate" means a complex or aggregate formed by one or more molecules of a solute, i.e. a compound or a pharmaceutically acceptable salt thereof, and one or more molecules of a solvent. Such solvates are typically crystalline solids having a substantially fixed molar ratio of solute and solvent. Representative solvents include, by way of example, water, methanol, ethanol, isopropanol, acetic acid and the like. When the solvent is water, the solvate formed is a hydrate.

The term "therapeutically effective amount" means an amount sufficient to effect treatment when administered to a patient in need of treatment, prevention or inhibition of a condition.

The term "patient" or "subject" refers to any vertebrate including, without limitation, humans and other primates (e.g., chimpanzees and other apes and monkey species), domestic mammals (e.g., dogs and cats), farm animals (e.g., cattle, sheep, pigs, goats and horses), laboratory animals (e.g., rodents such as mice, rats, and guinea pigs), and birds (e.g., domestic, wild and game birds such as chickens, turkeys and other gallinaceous birds, ducks, geese, and the like). In some embodiments, the subject is a mammal. In other embodiments, the subject is a human.

The present invention includes compounds, compositions and methods of

administering compositions to prevent, inhibit and/or treat bacterial infection, HIV infection, and cancer, and methods to promote stem cell mobilization, wound healing, hematopoiesis, and/or skin rejuvenation.

The compounds of the present invention may have, for example, the following structure:

or a pharmaceutically-acceptable salt or ester thereof, wherein:

Ri is selected from the group consisting of: hydrogen and

R₂ is selected from the group consisting

and

wherein each instance of D is independently selected from the group consisting of: hydrogen, linear or branched Ci-C₆ alkyl, linear or branched C₂-C₆ alkenyl, linear or branched C₂-C₆ alkynyl, and

wherein the linear or branched alkyl, alkenyl or alkynyls may include one or more spacer moieties selected from the group consisting of O, S, NH(C=0), (C=0)NH, 0(C=0), (C=0)0 or (C=0); each instance of A is independently selected from the group consisting of: linear or branched Ci-Ci₂ alkyl, linear or branched C₂-Ci₂ alkenyl, or linear or branched C₂-Ci₂ alkynyl, wherein the linear or branched alkyl, alkenyl or alkynyls may include one or more spacer moieties selected from the group consisting of: O, S, NH(C=0), (C=0)NH, 0(C=0), (C=0)0 or (C=0); each instance of R is independently selected from the group consisting of: hydrogen, Ci-C₆ alkyl, -NH₂,

III.

wherein phenyl, napthalene, and the monocyclic or bicyclic heteroaromatic group are each optionally substituted with one or more substituents independently selected from the group consisting of halo, hydroxy, alkyl, linear or branched Ci-C₆ alkyl, linear or branched C₂-C₆ alkenyl, linear or branched C₂-C₆ alkynyl, cycloalkyl, haloalkyl, hydroxyalkyl, alkoxy and alkoxyalkyl. In another specific embodiment, compounds may include the proviso that at least one R must be one of structures I-III.

In another specific embodiment of structure X, each instance of R may be independently selected from the group consisting of: -NH₂, structures I - II, and the following structure:

In another specific embodiment, structure X may be selected from the group consisting of:



and pharmaceutically acceptable salts or esters thereof. In a specific embodiment, each instance of R may be independently selected from the group consisting of: -NH₂ structures I- II, and the following structure:

The compounds of the present invention may also have, for example, the following structure:

or a pharmaceutically-acceptable salt or ester thereof, wherein:

Z is selected from the group consisting of straight chained or branched C_\-Cn alkyl, straight chained or branched C₂-C₁₂ alkenyl, straight chained or branched C₂-C₁₂ alkynyl; wherein the straight chained or branched alkyl, alkenyl or alkynyls may include one or more spacer moieties selected from the group consisting of O, S, NH(C=0), (C=0)NH, 0(C=0), (C=0)0, (C=0) and

IA.

IIA.

IIIA.

wherein the phenyl, napthalene, and the monocyclic or bicyclic heteroaromatic group are each optionally substituted with one or more substituents independently selected from the group consisting of halo, hydroxy, linear or branched Ci-C₆ alkyl, linear or branched C₂-C6 alkenyl, linear or branched C₂-C₆ alkynyl, cycloalkyl, haloalkyl, hydroxyalkyl, alkoxy and alkoxyalkyl;

and each instance of R is independently selected from the group consisting of:

hydrogen, -CH₃, -NH₂,

III.

wherein M

is selected from the group consisting of phenyl, napthalene, and a monocyclic or bicyclic heteroaromatic group, wherein the phenyl, napthalene, and the monocyclic or bicyclic heteroaromatic group are each optionally substituted with one or more substituents independently selected from the group consisting of halo, hydroxy, linear or branched Ci-C₆ alkyl, linear or branched C₂-C6 alkenyl, linear or branched C₂-C₆ alkynyl, cycloalkyl, haloalkyl, hydroxyalkyl, alkoxy and alkoxyalkyl;

and

In a specific embodiment, structure XI has the proviso that when Z does not include at least one spacer moiety that is

In another specific embodiment of structure XI, each instance of R may be independently selected from the group consisting of -NH₂, structures I - II and the following structure;

or a pharmaceutically acceptable salt or ester thereof; and wherein if said Z includes a spacer moiety which includes

each instance of Y is independently selected from the group consisting of hydrogen, structures IA - II A and the following structure:

In another specific embodiment, structure XI may be:

or pharmaceutically acceptable salts or esters thereof. In another specific embodiment, each instance of R may be independently selected from the group consisting of -NH₂, structures I - II and the following structure:

and each instance of Y is independently selected from the group consisting of hydrogen, structures I A - II A and the following structure:

In another specific embodiment, structure XI may be selected from the group consisting of

wherein R and Y comprise 2 structures independently selected from the group consisting of:

and

In another specific embodiment, R and Y may comprise 3 or more structures independently selected from the group consisting of:

In another embodiment, the present invention is directed to a pharmaceutically acceptable salt or ester or solvate or stereoisomer of the compounds provided above.

The compounds of the present invention may also contain one or more chiral centers and therefore, this invention is directed to racemic mixtures; pure stereoisomers (i.e., enantiomers or diastereomers); stereoisomer-enriched mixtures and the like unless otherwise indicated. When a particular stereoisomer is shown or named herein, it will be understood by those skilled in the art that minor amounts of other stereoisomers may be present in the compositions of this invention unless otherwise indicated, provided that the desired utility of the composition as a whole is not eliminated by the presence of such other isomers.

The compounds of the present invention may also contain several basic groups (e.g., amino groups) and therefore, the compounds of the present invention may exist as the free base or in various salt forms. All such salt forms are included within the scope of this invention. Furthermore, solvates of compounds of the present invention, or esters, or salts thereof are included within the scope of this invention.

Additionally, where applicable, all cis-trans or E/Z isomers (geometric isomers), tautomeric forms and topoisomeric forms of the compounds of the present invention are included within the scope of this invention unless otherwise specified.

The compounds of the present invention, as well as those compounds used in its synthesis, may also include isotopically-labeled compounds, i.e., where one or more atoms have been enriched with atoms having an atomic mass different from the atomic mass predominately found in nature. The present invention involves new therapeutics with novel mechanisms of action, which are needed for treatment of human immunodeficiency virus type 1 (HIV-1) infections. Maraviroc, a small molecule antagonist of the CCR5 receptor was the first drug of its type to be approved, and it has proven to be effective against "R5" HIV that uses the CCR5 co- receptor (9). However, most patients that have failed conventional therapies also harbor "X4" HIV that uses CXCR4 or dual-tropic viruses that can use either CCR5 or CXCR4. The appearance of the X4 viruses is associated with progression to AIDS symptoms (4), and drug resistance is more often linked to CXCR4-utilizing than to CCR5 -utilizing HIV (39).

Therefore, there is also a need to develop effective drugs that block CXCR4. So far, none of the CXCR4 antagonists in development have shown promise in HIV clinical trials, though some are being pursued as cancer therapeutics because CXCR4 may be involved in metastasis. Some CXCR4 inhibitors, such as the bicyclam AMD3100 and the monocyclam AMD3465, bind to a site located in the transmembrane domain of the receptor (13, 15, 26, 27, 30, 40). Like the CCR5-specific small molecule inhibitors, these compounds are believed to block viral entry and chemokine signaling by allosteric mechanism(s). In contrast to CCR5 inhibitors, however, many of the CXCR4-specific inhibitors contain multiple positive charges which can interact with additional acidic residues in the extracellular loops of CXCR4 that are not conserved in CCR5 (2, 10, 27, 28, 35).

CXCR4 and CCR5 belong to a group of 19 known chemokine G-protein coupled receptors. Ten of these chemokine receptors, including CCR5, belong to the CCR family and are numbered CCRl through CCR10. Seven chemokines, including CXCR4, belong to the CXCR family and are numbered CXCR1 through CXCR7. Two additional chemokines have been designated XCR1 and CX3CR1. CXCR4 and CCR5 have been shown to play a significant role in HIV infection. In some embodiments, the compounds of the present invention bind one or more chemokine receptors. In another specific embodiment, the chemokine receptor may be a G-protein coupled receptor. In another embodiment the G- protein coupled receptors may be selected from one or more of the group consisting of CCRl, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CXCR7, XCR1 and CX3CR1. In other embodiments, the compounds of the invention may bind the G-protein coupled receptors listed above. In a specific, emobiments, the compounds of the present invention may bind CXCR4. In other embodiments, the compounds of the present invention may bind CXCR4 without binding CCR5. In a specific embodiment, the compounds of the present invention may provide one or more of the following functions: a cytokine antagonist; improve skin rejuvenation; increase stem-cell mobilization; increase hematopoiesis; improve wound healing; treat, inhibit and/or prevent HIV; treat, inhibit and/or prevent cancer; and to treat, inhibit and/or prevent a viral infection.

In another specific embodiment, these functions may be the result of the compounds binding one or more active sites. In a specific embodiment, the one or more active sites may be a receptor, or specifically, a G-protein coupled receptor. In a specific embodiment, the receptor may be selected from one or more of the group consisting of CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CXCR7, XCR1 and CX3CR1.

For example, in a specific embodiment, the compound may bind a G-protein coupled receptor to treat, inhibit and/or prevent HIV. T-140, for example, is a 14-residue peptide antagonist of CXCR4 that blocks binding of X4 strains of HIV- 1 in vitro and displaces the natural agonist ligand SDF-1 a with nanomolar affinity but does not bind to other chemokine receptors, such as CCR5 (30-32). It is much smaller than SDF-la (14 residues vs 65 residues), but is rapidly degraded by proteolysis in serum (30) and has not been pursued as an HIV or cancer therapeutic. Recently, smaller cyclic peptides based on the structure of T-140 (25, 37), as well as non-peptide analogs (36), have been reported. However, the latter have IC₅₀S at least two orders of magnitude higher than that of T-140. The structure of T-140 contains 5 Arg and 2 Lys residues; the latter can be substituted with uncharged sidechains without loss of activity. ALX40-4C (N-alpha-acetyl-nona-D-arginine amide acetate) is another peptide antagonist of CXCR4 (8). An early clinical trial showed that it was well tolerated (7) and provided an important proof of principle that CXCR4 activity can be safely inhibited in humans. KRH-1636 is a CXCR4 antagonist which exhibits strong activity against X4 strains of HIV and competitively displaces SDF-1 from the receptor, and can be absorbed from the duodenum of rats (17). Like T-140 and ALX40-4C, KRH-1636 contains an Arg residue. An analog of this compound (KRH-2731) was reported to have enhanced activity and was claimed to be orally bioavailable in rats (24), but neither its structure nor any clinical trial results have been published. AMD3100 (plerixafor) is a bicyclam with 8 secondary and tertiary amine groups (5, 6). Clinical trials for HIV have been abandoned because this compound showed poor efficacy, cardiotoxicity, and it was not orally bioavailable (16). The structurally similar monocylam AMD3465 has higher affinity for CXCR4, and is potent against X4 HIV strains in vitro (IC₅₀: 1-10 nM) (19), but is also not orally bioavailable (14). AMD070 (a.k.a. AMD 11070), which has two aromatic rings in addition to a primary and a tertiary amine, has an IC₅₀ of 2-26 nM against an X4 HIV strain and is orally bioavailable (22, 29).

One objective of the present invention was to develop a series of discrete, small molecule inhibitors containing multiple guanide or biguanide groups and to test their ability to inhibit HIV-1 binding to CXCR4. Thus, in certain embodiments, a compound of the present invention may bind to a G-protein coupled receptors such as those that act as co- receptors for HIV-1 infection. In another embodiment, a compound of the present invention binds the G-protein coupled receptor(s) CXCR4 and/or CCR5. In another embodiment, a compound of the present invention binds CXCR4 with an IC₅₀ less than about 500 nM, about 450 nM, about 400 nM, about 350 nM, about 300 nM, about 250 nM, about 200 nM, about 150 nM, about 100 nM, about 50 nM, about 25 nM about 10 nM, about 5 nM, about 2.5 nM, or about 1 nM. In one embodiment, a compound of the present invention binds CXCR4 with an IC₅₀ less than about 500 μΜ, about 450 μΜ, about 400 μΜ, about 350 μΜ, about 300 μΜ, about 250 μΜ, about 200 μΜ, about 150 μΜ, about 100 μΜ, about 50 μΜ, about 25 μΜ, about 10 μΜ, about 5 μΜ, about 2.5 μΜ or about 1 μΜ in competition cross-link inhibition assays with T-140.

In some embodiments, the compounds of the present invention contain multiple guanide, biguanide, and/or arylguanide groups (such as phenylguanide groups) on linear aliphatic or dendrimer scaffolds. These compounds bear some resemblance to the CXCR4 antagonist peptide T-140, which has five guanide groups on the sidechains of arginine residues. Among the compounds of the present invention, the bis- and trisphenylguanide derivatives of spermidine exhibit the highest affinity for CXCR4, with an IC₅₀ of 200 nM in competition assays with T-140. This is comparable to that of other non-peptide analogs of T- 140 that have been reported (Ueda et al, (2008) Bioorg Med Chem Lett 18:4124-9), though it does not approach the nanomolar affinity of T-140 itself.

In other embodiments, the compounds of the present invention are spermine and spermidine derivatives. These spermine phenylguanide and spermidine phenylguanide are effective at inhibiting the infection of TZM-bl cells by X4, but not R5, HIV strains, consistent with the observation that they bind to CXCR4 but not CCR5. In fact, none of the compounds in Table 3 inhibited the binding of a fluorescent maraviroc analog to CCR5 (data not shown).

In one embodiment, the compound of the present invention is spermidine

phenylguanide, which has an IC₅₀ value of 3 μΜ when tested for inhibition of three X4 strains (NL4-3, 92HT599, and MN). In another embodiment, the compounds are underivatized parent amines of spermidine phenylguanide that show inhibition against both X4 and R5 strains of HIV, even though they are not effective in inhibiting T140 binding and cross-linking to CXCR4, suggesting that they act via another mechanism. In another embodiment, the compounds are underivatized DNT dendrimers that show, for example, antibacterial activity. These underivatized DNT dendrimers may be used to prevent, inhibit, and/or treat a bacterial infection in a patient in need thereof.

The optimal structure for a non-peptide CXCR4 antagonist cannot be readily predicted from the data available. Attempts to develop small cyclic peptide and non-peptide analogs from T-140 (Fujii et al, (2003) Angew Chem Int Ed Engl 42:3251-3; Ueda et al, (2008) Bioorg Med Chem Lett 18:4124-9; Ueda et al, (2007) J Med Chem 50: 192-8) have pointed to the importance of both a guanide functional group (in the form of an arginine sidechain) and an aromatic group (napthyl). This is consistent with our observation that the phenylguanides are more active than either the guanides or biguanides and suggests that using a larger aromatic moiety such as naphthalene may increase the observed CXCR4 binding compared to our initial phenylguanide derivatives.

The positioning of the aromatic group also appears to be important. For example, the most active phenylguanides (spermidine & spermine derivatives), with the aromatic groups on the ends and sides of the main chain of the molecule have at least an order of magnitude better binding than the best melamine derivative (THAM trisguanide), which has the aromatic group in the center of the molecule with the charges surrounding it. There also appears to be a necessary balance between charge and hydrophobic character. This is exemplified in the series of melamine derived phenylguanides in which increasing the length of the spacers between the melamine and phenylguanide groups, which increases overall hydrophobicity, led to a progressive loss of activity.

The spacing between the positively charged groups, at least in the linear compounds, also appears to be important. In the experiments leading to the present invention, the most active core molecules (spermine and spermidine) have spacings of three or four carbons between the reactive amine groups, whereas the less active diethylenetriamine and hexanediamine cores have two and six carbons respectively. However, the flexibility of the polyamine backbones versus those of the polybiguanides makes comparison of the distances between the charged groups in the two types of molecules difficult.

In addition to having antiviral activity, the guanide, biguanide, phenylguanide and arylguanide compounds of the present invention may also be a cytokine antagonist; improve skin rejuvenation; increase stem-cell mobilization; increase hematopoiesis; improve wound healing; treat, inhibit and/or prevent cancer; treat, inhibit and/or prevent a bacterial infection; and treat, inhibit and/or prevent a viral infection;.

In general, bacterial pathogens may be classified as either gram-positive or gram- negative pathogens. Antibiotic compounds with activity against both gram-positive and gram-negative pathogens are generally regarded as having a broad spectrum of activity. The compounds of the present invention are regarded as active against gram-positive and/or gram- negative bacterial pathogens. In particular, the present compounds are active against at least one gram-positive bacterium, preferably against several gram-positive bacteria, more preferably against one or more gram-positive bacteria and/or one or more gram-negative bacteria.

The compounds of the present invention have bactericidal or bacteriostatic activity. Examples of gram-positive and gram-negative aerobic and anaerobic bacteria, include Staphylococci, for example S. aureus; Enterococci, for example E. faecalis; Streptococci, for example S. pneumoniae, S. mutans, S. pyogens; Bacilli, for example Bacillus subtilis;

Listeria, for example Listeria monocytogenes; Haemophilus, for example H. influenza;

Moraxella, for example M. catarrhalis; Pseudomonas, for example Pseudomonas

aeruginosa; and Escherichia, for example E. coli.

Gram-positive pathogens, for example Staphylococci, Enterococci and Streptococci are particularly important because of the development of resistant strains which are both difficult to treat and difficult to eradicate from for example a hospital environment once established. Examples of such strains are methicillin resistant Staphylococcus aureus

(MRSA), methicillin resistant coagulase negative staphylococci (MRCNS), penicillin resistant Streptococcus pneumoniae and multiple resistant Enterococcus faecium.

The compounds of the present invention also show activity against resistant bacterial strains.

In one embodiment, the compounds of the present invention may be used to, inhibit, treat, and/or prevent a bacterial infection resulting from an infection of Acinetobacter baumannii, Burkholderia Cepacia, Enterococcus faecalis, Escherichia coli, Pseudomonas aeruginosa and/or Staphylococcus aureus. In another embodiment, the compounds of the present invention may be used to treat an infection of methicillin-resistant Staphylococcus aureus (MRSA).

In another embodiment of the present invention, the compounds of the present invention may be used to inhibit, treat, and/or prevent cancer. In a specific embodiment, the cancer may be selected from one or more of the group consisting of melanoma, leukemia, breast cancer, ovarian cancer, lung cancer, mesenchymal cancer, colon cancer, aerodigestive tract cancer, cervical cancer, brain tumors and prostate cancer.

The compounds of the present invention may be useful in the chemoprevention of cancer either alone or in combination with other anticancer agents. Chemoprevention is defined as inhibiting the development of invasive cancer by either blocking the initiating mutagenic event or by blocking the progression of pre-malignant cells that have already suffered an insult or inhibiting tumor relapse.

Cancer metastasis is a critical factor affecting the life expectancy of patients. It is reported that the expression of CXCR4 is enhanced in breast cancer cells, etc., and that the expression of CXCL12/SDF-l , which is a ligand of CXCR4, is enhanced in cancer- metastasized organs (lymph nodes, lungs, livers and bones). See Nature, Vol. 410, pp. 50-56 (2001). The compounds of the present invention may be useful in inhibiting tumor angiogenesis, neovascularization, and metastasis. As used herein, the term "metastasis" refers to the spread to other locations in the body, for example to another non-adjacent organ or part of an organ. Neovascularization is the formation of functional microvascular networks with red blood cell perfusion. The ability of CXCR4 antagonists to inhibit neovascularization is described further in U.S. Patent Publication 20040209837.

Angiogenesis is a process that is mainly characterized by the protrusion and outgrowth of capillary buds and sprouts from pre-existing blood vessels. Advantageously, especially the risk of tumor metastasis can be prevented or reduced by administering a compound of the present invention to a patient. The present invention provides preventive and/or therapeutic compounds that act as CXCR4 antagonists for cancers. The compounds of the present invention may show anti-cancer activity by antagonistically inhibiting the interaction of CXCR4 and its physiological ligand CXCL12/SDF-l . (e.g. migration inhibitory activity, invasion inhibitory activity, and anti-metastasis activity, etc.).

The term "anticancer" agent includes any known agent that is useful for the treatment of cancer including 17a-Ethinylestradiol, Diethylstilbestrol, Testosterone, Prednisone, Fluoxymesterone, Dromostanolone propionate, Testolactone, Megestrolacetate,

Methylprednisolone, Methyl-testosterone, Prednisolone, Triamcinolone, chlorotrianisene, Hydroxyprogesterone, Aminoglutethimide, Estramustine, Medroxyprogesteroneacetate, Leuprolide, Flutamide, Toremifene, Zoladex, matrix metalloproteinase inhibitors, VEGF inhibitors, including as anti-VEGF antibodies such as Avastin, and small molecules such as ZD6474 and SU6668, vatalanib, BAY-43-9006, SU11248, CP-547632, and CEP-7055 are also included. Anti-Her2 antibodies from Genentech (such as Herceptin) may also be utilized. Suitable EGFR inhibitors include gefitinib, erlotinib, and cetuximab. Pan Her inhibitors include canertinib, EKB-569, and GW-572016. Also included are Src inhibitors as well as Casodex® (bicalutamide, Astra Zeneca), Tamoxifen, MEK-1 kinase inhibitors, MAPK kinase inhibitors, PI3 inhibitors, and PDGF inhibitors, such as imatinib. Also included are anti- angiogenic and antivascular agents which, by interrupting blood flow to solid tumors, render cancer cells quiescent by depriving them of nutrition. Castration, which also renders androgen dependent carcinomas non-proliferative, may also be utilized. Also included are IGFIR inhibitors, inhibitors of non-receptor and receptor tyrosine kinases, and inhibitors of integrin signaling. Additional anticancer agents include microtubule-stabilizing agents such as paclitaxel (also known as Taxol®), docetaxel (also known as Taxotere®), 7-0- methylthiomethylpaclitaxel (disclosed in U.S. Pat. No. 5,646,176), 4-desacetyl-4- methylcarbonatepaclitaxel, 3 '-tert-buty 1-3 '-N-tert-butyloxycarbonyl-4-deacetyl-3 ' -dephenyl- 3'-N-debenzoyl-4-0-methoxycarbonyl-paclitaxel (disclosed in U.S. Ser. No. 09/712,352 filed on Nov. 14, 2000), C-4 methyl carbonate paclitaxel, epothilone A, epothilone B, epothilone C, epothilone D, desoxyepothilone A, desoxyepothilone B, [1S-[1R*, 3R*(E), 7R*, 10S*, 11R*, 12R*, 16S*]]-7-l l-dihydroxy-8,8 ,10,12,16-pentamethyl-3-[l-methyl-2-(2-methyl-4- thiazolyl)et henyl]-4-aza-17 oxabicyclo[14.1.0]heptadecane-5,9-dione (disclosed in WO 99/02514), [1S-[1R*, 3R*(E), 7R*, 10S*, 11R*, 12R*, 16S*]]-3-[2-[2-(aminometh yl)-4- thiazolyl]- 1 -methylethenyl]-7, 11 -dihydroxy-8,8, 10,12,1 6-pentamethyl-4-l 7- dioxabicyclo[14.1.0]-heptadecane-5,9-dion e (disclosed in U.S. Pat. No. 6,262,094) and derivatives thereof; and microtubule-disruptor agents. Also suitable are CDK inhibitors, an antiproliferative cell cycle inhibitor, epidophyllotoxin; an antineoplastic enzyme; a topoisomerase inhibitor; procarbazine; mitoxantrone; platinum coordination complexes such as cisplatin and carboplatin; biological response modifiers; growth inhibitors; antihormonal therapeutic agents; leucovorin; tegafur; and haematopoietic growth factors.

Additional cytotoxic agents include, melphalan, hexamethyl melamine, thiotepa, cytarabin, idatrexate, trimetrexate, dacarbazine, L-asparaginase, camptothecin, topotecan, bicalutamide, flutamide, leuprolide, pyridobenzoindole derivatives, interferons, and interleukins.

In chronic rheumatoid arthritis, the infiltration of CD4 positive memory T-cells into articular cavity fluids affects the progression of the disease. It is reported that in CD4 positive T-cells in articular cavity fluids of patients suffering from chronic rheumatoid arthritis, the expression of CXCR4 genes is enhanced, and that the expression of CXCL12/SDF-l genes is enhanced in articular synovial membrane tissues. See Journal of Immunology, Vol. 165, pp. 6590-98 (2000). In some embodiments, the compounds of the present invention may be used to for the prevention and/or therapy of chronic rheumatoid arthritis. In these

embodiments, the compounds may have an inhibitory effect on T-cell movement.

In some embodiments, the compounds of the invention may be useful in modulating chemokine-receptor signaling and stem cell trafficking. The SDF-1/CXCR4 axis emerges as a pivotal regulator of trafficking of various types of stem cells in the body. Since most if not all malignancies originate in the stem/progenitor cell compartment, cancer stem cells also express CXCR4 on their surface and, as a result, the SDF-1/CXCR4 axis is involved in directing their trafficking/metastasis to organs that express SDF-1 such as, e.g., lymph nodes, lungs, liver, and bones. In consequence, strategies aimed at modulating the SDF-1/CXCR4 axis have important clinical applications both in regenerative medicine to deliver normal stem cells to the tissues and in clinical oncology to inhibit metastasis of cancer stem cells. See Kucia, M., R. Reca, et al. (2005). "Trafficking of normal stem cells and metastasis of cancer stem cells involve similar mechanisms: pivotal role of the SDF-1-CXCR4 axis." Stem Cells 23(7): 879-94.

In another embodiment, the compounds of the present invention may be used to promote stem cell mobilization. Leukocytes, also known as white blood cells, include neutrophils, macrophages, eosinophils, basophils/mast cells, B cells and T cells. White blood cells are continuously replaced via the hematopoietic system, by the action of colony stimulating factors (CSFs) and various cytokines on stem cells and progenitor cells in hematopoietic tissues. The most widely known of these factors is granulocyte colony stimulating factor (abbr. G-CSF) which has been approved for use in counteracting the negative effects of chemotherapy by stimulating the production of white blood cells and progenitor cells (peripheral blood stem cell mobilization). There are a number of cell surface antigens that are used as markers for the characterization of the stem and progenitor cell populations. These markers are also subject to change, whenever new, more specific markers are discovered. Hematopoietic stem cells are currently characterized by being CD34+, c-kit+, Sca-1+, CD45+, lin-, and CD38- (CD 38 is also a lineage marker, this is therefore redundant to lid) The bone marrow is also a host for several other stem cell types that are not hematopoietic, but may give rise to other cell types and tissues: Mesenchymal stem cells are characterized as CD34+, Sca-1+, lin-, BMPR+ and/or STRO-1+, tissue-committed stem cells from bone marrow: are currently defined as being CXCR4+, CD34+, CD45-. Subpopulations of the tissue-committed stem cells from bone marrow are: skeletal stem cells: Myf5+, MyoD+; cardiac stem cells: NKx2.5+, GATA4+; liver stem cells: CK19+, a-fetoprotein+; and neural stem cells: nestin+, GATA4+. See Majka M, Kucia M, Ratajczak M Z (2005) Stem cell biology— a never ending quest for understanding. Acta Biochim Pol 52(2): 353- 358.

Several other factors have been reported to increase white blood cells and progenitor cells in both human and animal subjects. These agents include granulocyte-macrophage colony stimulating factor (abbr. GM-CSF), Interleukin-1 (abbr. IL-1), Interleukin-3 (abbr. IL- 3), Interleukin-8 (abbr. IL-8), PIXY-321 (abbr. GM-CSF/IL-3 fusion protein), macrophage inflammatory protein (abbr. MIP), GRCf (CXCL2) and GRCfT(CXCL2A4), stem cell factor, thrombopoietin and growth related oncogene, as single agents or in combination. See, e.g., King, A. G., D. Horowitz, et al. (2001). "Rapid mobilization of murine hematopoietic stem cells with enhanced engraftment properties and evaluation of hematopoietic progenitor cell mobilization in rhesus monkeys by a single injection of SB-251353, a specific truncated form of the human CXC chemokine GRObeta." Blood 97(6): 1534-42. Pruijt, J. F., R.

Willemze, et al. (1999). "Mechanisms underlying hematopoietic stem cell mobilization induced by the CXC chemokine interleukin-8." Curr Opin Hematol 6(3): 152-8.

While endogenous growth factors are pharmacologically effective, the well known disadvantages of employing proteins and peptides as pharmaceuticals underline the need to add to the repertoire of such growth factors further agents which are effective insofar, i.e. which increase progenitor cells of leukocytes and stem cells, respectively, preferably increase the level thereof in peripheral blood of a subject. Accordingly, one objective of the present invention is to provide means and methods for increasing progenitor cells of leukocytes and stem cells, respectively. In one embodiment, the administration of the compounds of the present invention may specifically increase the level of progenitor cells of leukocytes and/or stem cells in peripheral blood of a subject. A further objective of the present invention is to provide means and methods for the treatment of diseases which are caused by or associated with low level of progenitor cells of leukocytes and stem cells, respectively.

Stem cells are either mobilized in order to directly enable the repair of damaged tissues in the same patient in which they are mobilized, or they are mobilized and collected from a human leukocyte antigen (HLA) matched donor and administered to the patient either intra venously or directly into an affected tissue. The latter can also be done with stem cells that were mobilized from the patient himself. Before administration of the stem cells, they can be expanded and/or differentiated in vitro.

AMD3100 (plerixafor) is an approved drug for mobilization of stem cells that acts by binding to CXCR4. Aminoglycoside-polyarginine conjugates also bind CXCR4 and have been show to act synergistically with AMD3100 for this application. See Berchanski A, Kalinkovich A, Ludin A, Lapidot T, Lapidot A., (2011) Insight into the mechanism of enhanced mobilization of hematopoietic progenitor cells and release of CXCL12 by combination of AMD3100 and aminoglycoside-polyarginine conjugates,

FEBS J. 2011 Sep 12. doi: 10.1111/j.1742-4658.2011.08348.x. [Epub ahead of print]. In certain embodiments, the compounds of the present invention bind CXCR4 and act to mobilize stem cells. In other embodiments, compounds of the present invention with high affinity for CXCR4 may work synergistically with AMD3100 and have advantages over the polyarginine conjugates in stem cell mobilization. In another embodiment, the compounds of the present invention may be used in combination with other factors that have been reported to increase white blood cells and progenitor cells in patients.

In other embodiments, the compounds of the present invention may form a composition with one or more pharmaceutically acceptable excipients. In another

embodiment, the invention is a method of administering an effective amount of one of the compositions of the present invention to a patient in need thereof. In one embodiment the patient is in need of a composition comprising the compounds that provide a function as a cytokine antagonist; to improve skin rejuvenation; to increase stem-cell mobilization; to increase hematopoiesis; to improve wound healing; to treat, inhibit and/or prevent HIV; to treat, inhibit and/or prevent cancer; to treat, inhibit and/or prevent a bacterial infection; and to treat, inhibit and/or prevent a viral infection.

In one embodiment, the invention is a method of administering an effective amount of one of the compounds of the present invention to a patient that has been previously diagnosed with a bacterial infection, an HIV infection, and/or cancer. In another embodiment, the administration of a compound of the present invention to a patient results in a decrease of the bacterial population, the HIV viral load, and/or the growth or metastasis of cancer cells in the patient as compared to those levels in the patient before administration of the compound. In another embodiment, the compounds of the present invention may be used to treat an HIV infection resulting from an infection by the R5 and/or X4 strain(s) of HIV- 1.

The pharmaceutical compositions containing the compounds of the present invention may be in a form suitable for oral use, for example, as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules, or syrups or elixirs. Compositions intended for oral use may be prepared according to any method known to the art for the manufacture of pharmaceutical compositions and such compositions may contain one or more agents selected from the group consisting of sweetening agents, flavoring agents, coloring agents and preserving agents in order to provide pharmaceutically elegant and palatable preparations. Tablets contain the compounds of the present invention in admixture with non-toxic pharmaceutically acceptable excipients which are suitable for the manufacture of tablets. These excipients may be for example, inert diluents, such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, for example, microcrystalline cellulose, sodium crosscarmellose, corn starch, or alginic acid; binding agents, for example starch, gelatin, polyvinyl-pyrrolidone or acacia, and lubricating agents, for example, magnesium stearate, stearic acid or talc. The tablets may be uncoated or they may be coated by known techniques to mask the unpleasant taste of the drug or delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a water soluble taste masking material such as hydroxypropyl-methylcellulose or hydroxypropyl-cellulose, or a time delay material such as ethyl cellulose, cellulose acetate buryrate may be employed.

Formulations for oral use may also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredient is mixed with water soluble carrier such as polyethyleneglycol or an oil medium, for example peanut oil, liquid paraffin, or olive oil.

Aqueous suspensions contain the compounds of the present invention in admixture with excipients suitable for the manufacture of aqueous suspensions. Such excipients are suspending agents, for example sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethyl-cellulose, sodium alginate, polyvinyl-pyrrolidone, gum tragacanth and gum acacia; dispersing or wetting agents may be a naturally-occurring phosphatide, for example lecithin, or condensation products of an alkylene oxide with fatty acids, for example polyoxyethylene stearate, or condensation products of ethylene oxide with long chain aliphatic alcohols, for example heptadecaethylene-oxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as

polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, for example polyethylene sorbitan monooleate. The aqueous suspensions may also contain one or more preservatives, for example ethyl, or n-propyl p-hydroxybenzoate, one or more coloring agents, one or more flavoring agents, and one or more sweetening agents, such as sucrose, saccharin or aspartame. Oily suspensions may be formulated by suspending the compounds of the present invention in a vegetable oil, for example arachis oil, olive oil, sesame oil or coconut oil, or in mineral oil such as liquid paraffin. The oily suspensions may contain a thickening agent, for example beeswax, hard paraffin or cetyl alcohol. Sweetening agents such as those set forth above, and flavoring agents may be added to provide a palatable oral preparation. These compositions may be preserved by the addition of an anti-oxidant such as butylated hydroxyanisol or alpha-tocopherol.

Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the active ingredient in admixture with a dispersing or wetting agent, suspending agent and one or more preservatives. Suitable dispersing or wetting agents and suspending agents are exemplified by those already mentioned above. Additional excipients, for example sweetening, flavoring and coloring agents, may also be present. These compositions may be preserved by the addition of an anti-oxidant such as ascorbic acid.

The pharmaceutical compositions of the invention may also be in the form of an oil- in- water emulsions. The oily phase may be a vegetable oil, for example olive oil or arachis oil, or a mineral oil, for example liquid paraffin or mixtures of these. Suitable emulsifying agents may be naturally-occurring phosphatides, for example soy bean lecithin, and esters or partial esters derived from fatty acids and hexitol anhydrides, for example sorbitan monooleate, and condensation products of the said partial esters with ethylene oxide, for example polyoxyethylene sorbitan monooleate. The emulsions may also contain sweetening, flavoring agents, preservatives and antioxidants.

Syrups and elixirs may be formulated with sweetening agents, for example glycerol, propylene glycol, sorbitol or sucrose. Such formulations may also contain a demulcent, a preservative, flavoring and coloring agents and antioxidant.

The pharmaceutical compositions may be in the form of a sterile injectable aqueous solutions. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution.

The sterile injectable preparation may also be a sterile injectable oil-in-water microemulsion where the active ingredient is dissolved in the oily phase. For example, the active ingredient may be first dissolved in a mixture of soybean oil and lecithin. The oil solution then introduced into a water and glycerol mixture and processed to form a microemulation. The injectable solutions or microemulsions may be introduced into a patient's bloodstream by local bolus injection. Alternatively, it may be advantageous to administer the solution or microemulsion in such a way as to maintain a constant circulating concentration of the instant compound. In order to maintain such a constant concentration, a continuous intravenous delivery device may be utilized. An example of such a device is the Deltec CADD-PLUS™ model 5400 intravenous pump.

The pharmaceutical compositions may be in the form of a sterile injectable aqueous or oleagenous suspension for intramuscular and subcutaneous administration. This suspension may be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents which have been mentioned above. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally- acceptable diluent or solvent, for example as a solution in 1,3-butane diol. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil may be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables.

The compounds of the present invention may also be administered in the form of a suppositories for rectal administration of the drug. These compositions can be prepared by mixing the drug with a suitable non-irritating excipient which is solid at ordinary

temperatures but liquid at the rectal temperature and will therefore melt in the rectum to release the drug. Such materials include cocoa butter, glycerinated gelatin, hydrogenated vegetable oils, mixtures of polyethylene glycols of various molecular weights and fatty acid esters of polyethylene glycol.

For topical use, creams, ointments, jellies, solutions or suspensions, etc., containing the compounds of the present invention are employed. (For purposes of this application, topical application shall include mouth washes and gargles.) For example, a topical formulation would be particularly effective for skin rejuvenation.

The compounds for the present invention can be administered in intranasal form via topical use of suitable intranasal vehicles and delivery devices, or via transdermal routes, using those forms of transdermal skin patches well known to those of ordinary skill in the art. To be administered in the form of a transdermal delivery system, the dosage administration will, of course, be continuous rather than intermittent throughout the dosage regimen.

Compounds of the present invention may also be delivered as a suppository employing bases such as cocoa butter, glycerinated gelatin, hydrogenated vegetable oils, mixtures of polyethylene glycols of various molecular weights and fatty acid esters of polyethylene glycol.

When a compound according to this invention is administered into a human subject, the daily dosage will normally be determined by the prescribing physician with the dosage generally varying according to the age, weight, sex and response of the individual patient, as well as the severity of the patient's symptoms.

If formulated as a fixed dose, such combination products employ the compounds of this invention within the dosage range described above and the other pharmaceutically active agent or treatment within its approved dosage range. The compounds of the present invention may also be administered sequentially with other known antibiotics and/or antiviral agents and/or anticancer or cytotoxic agents when a combination formulation is inappropriate. The invention is not limited in the sequence of administration; the compounds of the present invention may be administered either prior to or after administration of the other known antibiotics and/or antiviral agents and/or anticancer or cytotoxic agent(s).

The invention also encompasses methods where the compound is given in

combination therapy in treating AIDS and HIV infection. That is, the compound can be used in conjunction with, but separately from, other agents useful in treating AIDS and HIV infection. Some of these agents include HIV attachment inhibitors, CCR5 inhibitors, CXCR4 inhibitors, HIV cell fusion inhibitors, HIV integrase inhibitors, HIV nucleoside reverse transcriptase inhibitors, HIV non-nucleoside reverse transcriptase inhibitors, HIV protease inhibitors, budding and maturation inhibitors, immunomodulators, and anti-infectives. In these combination methods, the compounds of the present invention will generally be given in a daily dose of 1-100 mg/kg body weight daily in conjunction with other agents. The other agents generally will be given in the amounts used therapeutically. The specific dosing regime, however, will be determined by a physician using sound medical judgement.

Table 1 lists some agents useful in treating AIDS and HIV infection which are suitable for this invention. Table 1. Therapeutic agents for treatment of AIDS and HIV infection.

DRUG NAME MANUFACTURER INDICATION

ANTIVIRAL S

097 Hoechst/Bayer HIV infection, AIDS,

(non-nucleoside ARC

reverse

transcriptase

inhibitor)

Amprenavir Glaxo Wellcome HIV infection, AIDS,

141 W94 ARC

GW 141

(protease inhibitor)

Abacavir (1592U89) Glaxo Wellcome HIV infection, AIDS, GW 1592 ARC

(RT inhibitor)

Acemannan Carrington Labs ARC

(Irving, TX)

Acyclovir Burroughs Wellcome HIV infection, AIDS,

ARC, in combination with AZT

AD-439 Tanox Biosystems HIV infection, AIDS,

ARC AD-519 Tanox Biosystems HIV infection, AIDS,

ARC

Adefovir dipivoxil Gilead Sciences HIV infection, ARC, AL-721 Ethigen PGL HIV positive, AIDS (Los Angeles, CA)

Alpha Interferon Glaxo Wellcome Kaposi's sarcoma HIV in combination

withRetrovir

Ansamycin Adria Laboratories ARC

LM 427 (Dublin, OH)

Erbamont

(Stamford, CT)

Antibody which Advanced Biotherapy AIDS, ARC

Neutralizes pH Concepts

Labile alpha aberrant (Rockville, MD)

Interferon

AR177 Aronex Pharm HIV infection, AIDS,

ARC

Beta-fluoro-ddA Nat'l Cancer Institute AIDS-associated diseases

BMS-232623 Bristol-Myers Squibb/ HIV infection, AIDS,

(CGP-73547) Novartis ARC

(protease inhibitor)

BMS-234475 Bristol-Myers Squibb/ HIV infection, AIDS,

(CGP-61755) Novartis ARC

(protease inhibitor)

CI-1012 Warner-Lambert HIV-1 infection

Cidofovir Gilead Science CMV retinitis, herpes, papillomavirus

Curdlan sulfate AJI Pharma USA HIV infection

Cytomegalovirus Medlmmune CMV retinitis

Immune globin

Cytovene Syntex Sight threatening Ganciclovir CMV peripheral, CMV retinitis

Delaviridine Pharmacia-Upj ohn HIV infection, AIDS, (RT inhibitor) ARC

Dextran Sulfate Ueno Fine Chem. AIDS, ARC, HIV

Ind. Ltd. (Osaka, positive asymptomatic Japan)

ddC Hoffman-La Roche HIV infection, AIDS,

Dideoxycytidine ARC

ddl Bristol-Myers Squibb HIV infection, AIDS,

Dideoxyinosine ARC; combinationwith

AZT/d4T

DMP-450 AVID HIV infection, AIDS, (protease inhibitor) (Camden, NJ) ARC

Efavirenz DuPont Merck HIV infection, AIDS, (DMP 266) ARC

(-)6-Chloro-4-(S)- cyclopropylethynyl- 4(S)-trifluoro- methyl- 1 ,4-dihydro- 2H-3 , 1 -benzoxazin- 2-one, STOCRINE

(non-nucleoside RT

inhibitor)

EL10 Elan Corp, PLC HIV infection

(Gainesville, GA)

Famciclovir Smith Kline herpes zoster, herpes simplex

FTC Emory University HIV infection, AIDS,

(reverse transcriptase ARC inhibitor)

GS 840 Gilead HIV infection, AIDS,

(reverse transcriptase ARC

inhibitor)

HBY097 Hoechst Marion HIV infection, AIDS,

(non-nucleoside Roussel ARC

reverse

transcriptase

inhibitor)

Hypericin VIMRx Pharm. HIV infection, AIDS,

ARC

Recombinant Human Triton Biosciences AIDS, Kaposi's sarcoma, Interferon Beta (Almeda, CA) ARC

Interferon alfa-n3 Interferon Sciences ARC, AIDS

Indinavir Merck HIV infection, AIDS,

ARC, asymptomatic HIV positive, also in combination with AZT/ddl/ddC

ISIS 2922 ISIS Pharmaceuticals CMV retinitis

KNI-272 Nat'l Cancer Institute HIV-associated diseases

Lamivudine, 3TC Glaxo Wellcome HIV infection, AIDS,

(reverse transcriptase ARC, also with AZT inhibitor)

Lobucavir Bristol-Myers Squibb CMV infection

Nelfmavir Agouron HIV infection, AIDS,

(protease inhibitor) Pharmaceuticals ARC

Nevirapine Boeheringer HIV infection, AIDS,

(RT inhibitor) Ingleheim ARC Novapren Novaferon Labs, Inc. HIV inhibitor

(Akron, OH)

Peptide T Peninsula Labs AIDS

Octapeptide (Belmont, CA)

Sequence

Trisodium Astra Pharm. CMV retinitis, HIV

Phosphonoformate Products, Inc. infection, other CMV infections

PNU- 140690 Pharmacia Upjohn HIV infection, AIDS, (protease inhibitor) ARC

Probucol Vyrex HIV infection, AIDS RBC-CD4 Sheffield Med. HIV infection, AIDS,

Tech (Houston, TX) ARC

Ritonavir Abbott HIV infection, AIDS,

(protease inhibitor) ARC

Saquinavir Hoffmann- HIV infection, AIDS,

(protease inhibitor) LaRoche ARC

Stavudine; d4T Bristol-Myers Squibb HIV infection, AIDS,

Didehydrodeoxy- ARC

thymidine

Valaciclovir Glaxo Wellcome Genital HSV &

CMVinfections

Virazole Viratek/ICN asymptomatic HIV-

Ribavirin (Costa Mesa, CA) positive, LAS, ARC

VX-478 Vertex HIV infection, AIDS,

ARC

Zalcitabine Hoffmann-LaRoche HIV infection, AIDS,

ARC, with AZT

Zidovudine; AZT Glaxo Wellcome HIV infection, AIDS, ARC, Kaposi's sarcoma, in combination with other therapies

Tenofovir disoproxil, Gilead HIV infection, AIDS fumarate salt

(Viread ®)

(reverse transcriptase

inhibitor)

Combivir ® GSK HIV infection, AIDS

(reverse transcriptase

inhibitor)

abacavir succinate GSK HIV infection, AIDS

(or Ziagen ®)

(reverse transcriptase

inhibitor)

Reyataz ® Bristol-Myers Squibb HIV infection, AIDS

(atazanavir)

Fuzeon Roche/Trimeris HIV infection, AIDS,

(Enfuvirtide, T-20) viral fusion inhibitor Trizivir ® HIV infection, AIDS Kaletra ® Abbott HIV infection, AIDS

ARC

IMMUNOMODULATORS

AS-101 Wyeth-Ayerst AIDS

Bropirimine Pharmacia Upjohn Advanced AIDS

Acemannan Carrington Labs, Inc. AIDS, ARC

(Irving, TX)

CL246,738 American Cyanamid AIDS, Kaposi's sarcoma

Lederle Labs EL10 Elan Corp, PLC HIV infection

(Gainesville, GA)

FP-21399 Fuki ImmunoPharm Blocks HIV fusion with

CD4+ cells

Gamma Interferon Genentech ARC, in combination withTNF (tumor necrosis factor)

Granulocyte Genetics Institute AIDS

Macrophage Colony Sandoz

Stimulating Factor

Granulocyte Hoechst-Roussel AIDS

Macrophage Colony Immunex

Stimulating Factor

Granulocyte Schering-Plough AIDS, combination Macrophage Colony withAZT

Stimulating Factor

HIV Core Particle Rorer Seropositive HIV

Immunostimulant

IL-2 Cetus AIDS, in combination

Interleukin-2 withAZT

IL-2 Hoffman-LaRoche AIDS, ARC, HIV, in

Interleukin-2 Immunex combination withAZT IL-2 Chiron AIDS, increase in CD4

Interleukin-2 cell counts

(aldeslukin)

Immune Globulin Cutter Biological Pediatric AIDS, in

Intravenous (Berkeley, CA) combination withAZT

(human)

IMREG-1 Imreg AIDS, Kaposi's sarcoma, (New Orleans, LA) ARC, PGL

IMREG-2 Imreg AIDS, Kaposi's sarcoma,

(New Orleans, LA) ARC, PGL

Imuthiol Diethyl Merieux Institute AIDS, ARC

Dithio Carbamate

Alpha-2 Schering Plough Kaposi's sarcoma Interferon withAZT, AIDS

Methionine- TNI Pharmaceutical AIDS, ARC

Enkephalin (Chicago, IL)

MTP-PE Ciba-Geigy Corp. Kaposi's sarcoma AIDS,

Muramyl-Tripeptide Amgen in combination withAZT

Granulocyte

Colony Stimulating

Factor

Remune Immune Response Immunotherapeutic

Corp.

rCD4 Genentech AIDS, ARC

Recombinant

Soluble Human CD4

rCD4-IgG AIDS, ARC

hybrids

Recombinant Biogen AIDS, ARC

Soluble Human CD4

Interferon Hoffman-La Roche Kaposi's sarcoma, AIDS,

Alfa 2a in combination withAZT ARC

SK&F 106528 Smith Kline HIV infection

Soluble T4

Thymopentin Immunobiology HIV infection

Research Institute (Annandale, NJ)

Tumor Necrosis Genentech ARC, in combination Factor; TNF withgamma Interferon ANTI-INFECTIVES

Clindamycin with Pharmacia Upjohn PCP

Primaquine

Fluconazole Pfizer Cryptococcal meningitis, candidiasis

Pastille Squibb Corp. Prevention of oral

Nystatin Pastille candidiasis

Ornidyl Merrell Dow PCP

Eflornithine

Pentamidine LyphoMed PCP treatment

Isethionate (IM & IV) (Rosemont, IL)

Trimethoprim Antibacterial

Trimethoprim/ sulfa Antibacterial

Piritrexim Burroughs Wellcome PCP treatment

Pentamidine Fisons Corporation PCP prophylaxis

Isethionate for

Inhalation

Spiramycin Rhone-Poulenc Cryptosporidial

diarrhea

Intraconazole- Janssen-Pharm. Histoplasmosis;

R5121 1 cryptococcal meningitis

Trimetrexate Warner-Lambert PCP

Daunorubicin NeXstar, Sequus Kaposi's sarcoma

Recombinant Human Ortho Pharm. Corp. Severe anemia assoc.

Erythropoietin with AZT therapy

Recombinant Human Serono AIDS-related wasting, Growth Hormone cachexia

Megestrol Acetate Bristol-Myers Squibb Treatment of anorexia

assoc. WITHAIDS

Testosterone Alza, Smith Kline AIDS-related wasting

Total Enteral Norwich Eaton Diarrhea and

Nutrition Pharmaceuticals malabsorption related to

AIDS

In some embodiments, the compounds of the present invention are provided as discrete, small molecule inhibitors of the binding and/or activation of the CXCR4 co- receptor. The compounds of the present invention, especially the phenylguanides, represent promising therapeutic agents as non-peptide inhibitors of X4 HIV infection. Their very low cytotoxicity makes them promising candidates for treatment of bacterial infection, HIV infection, and cancer, and their non-peptide structures gives them greater serum stability than other therapeutic agents such as T 140.

All publications, patents and patent applications, including any drawings and appendices, herein are incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be

incorporated by reference.

This invention is further illustrated by the following additional examples that should not be construed as limiting. Those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made to the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

EXAMPLES

Example 1

Guanide, biguanide, and phenylguanide synthesis. The results of the compound syntheses are summarized in Table 2. Starting material compounds (see Figure 1 for structures) are listed in the first column with the number of amines available for addition of guanide, biguanide, or phenylguanide groups (see Figure 2) in parentheses. The results for each column show the number of amine groups derivatized. In cases labeled with Or', two fractions were isolated with each of the respective products (e.g. both the monophenyl- guanide and bisphenylguanide additions to putrescine were isolated and tested separately). In cases labeled with a dash, the designated products were inseparable by HPLC (e.g. DNT2300 yielded a mixture of compounds with either 5 or 6 guanide groups per molecule).

Other deviations from the expected products are noted in Table 2. In three of the guanide addition reactions, additional +15 and sometimes +30 amu products were co-purified with the isolated products. For these compounds, 20-50% of the isolated product was observed to be the +15 product, with a small amount (<10%>) of the +30 product. These compounds were tested for activity as isolated; later, it was discovered that these products were the result of loss of ammonia during the substitution reaction instead of the expected loss of methanol. Treatment of these compounds with concentrated ammonium hydroxide can replace the O-methyl groups and yield the desired products, (see Figure 3), but the effect of this reaction on the activity of the compound was not tested. Some reactions with O- methylisourea yielded a moderate amount (20-50%) of a +15 amu byproduct. Formation of this product can be rationalized through the zwitterionic tetrahedral SN₂ intermediate eliminating NH₃, instead of the expected CH₃OH, to give the O-methylisourea byproduct. Treatment of this compound with concentrated NH₄OH yields the desired guanide.

The reaction of diethylenetriamine (DETA) with the phenylguanide reagent resulted in some cyclic byproducts (see Figure 4) that can be encountered when the reactive amine groups are closely spaced. Mass signals in the MALDI analysis of the DETA phenylguanide synthesis suggest some side reactions occurred. In the proposed mechanism, a nucleophilic amine with the correct spacing to form a five-membered ring intermediate attacks the phenylguanide imine, which can either revert to the bis-phenylguanide or eliminate ammonia or aniline to give both the byproduct masses observed by mass spectrometry. Similar byproducts were not observed with reactions of any of the other starting amine compounds. These same mechanisms are probably involved in the formation of the products labeled 'cyanoamine and biguanides' in the reaction of DETA with S-methyl-guanylisothiourea. This reaction proceeds through the loss of urea and the formation of a cyclic product, as seen with the phenylguanides, or to the formation of a cyanoamine. However, mass spectrometry is unable to differentiate between the two byproducts.

ND synthesis not done.

Example 2

Antimicrobial activity and cytotoxicity in vitro. The in vitro activity of all 48 compounds was tested against Acinetobacter baumannii (ATCC 19606, ATCC BAA1605), Enterococcus faecalis, Escherichia coli K12, Pseudomonas aeruginosa PAOl and

Staphylococcus aureus by testing each compound at 30 and 100 μΜ in a cell viability assay. To conduct the bacterial cell viability assays, each bacterial species was grown at 37°C to mid-log phase (OD₆oo -0.4). The bacteria were diluted 1/1000 and added to a solution of PBS containing the compound at multiple different concentrations. The mixture was incubated at 37°C for one hour and then plated in duplicate on rich media in 1 x PBS. The plates were incubated overnight at 37°C and colonies counted the next day. The IC₅₀s were calculated from graphs of colony forming units (CFU) as a function of compound concentration using SigmaPlot. Each IC₅₀ was determined at least twice. Chlorhexidine was used as a positive control for all assays. For all the guanide, biguanide, and phenylguanide compounds that showed significant inhibition at 30 μΜ, as well as for the parent amines from which they had been synthesized, we then determined the IC₅₀ using the same assay (Table 3). In this assay, the cells and compound are diluted after one hour of treatment and the bacteria are then allowed to grow on plates in the absence of compound. Therefore, the compounds were bactericidal at the concentrations that resulted in decreased CFUs.

The in vitro toxicity of the compounds to human cells was evaluated using an MTS dye reduction assay in both MDA-MB-231 and HaCaT cells, and the results are shown in Table 5.

Although the fact that some of our compounds were biguanides led us to infer a similarity to PHMB and chlorhexidine, many of our biguanides had less antibacterial activity than the corresponding guanides or phenylguanides. Therefore, it is likely that the active compounds we are pursuing act by different mechanisms than chlorhexidine or PHMB. A compound with four guanide groups, p-guanidinoethylcalix[4]arene, was reported to have antibacterial activity, but not the monomeric unit p-guanidinoethylphenol (Grare et al., (2010) Clin Microbiol Infect. 16:432-8). Our data suggest that both the multimeric structure and the functional group are important, with the phenylguanides being particularly active.

Attorney Docket No. MONT- 119/01 W

Table 3. Calculated values (±SEM) of the IC_5Q (μΜ) of each compound against the various microbial species. IC50S of those guanide

(G), biguanide (BG) and phenylguanide (OG) compounds that had some antibacterial activity in an initial filter disk screen against 5 bacterial pathogens, and IC50S of the parent amines are shown. Depending on the compound and counterion, ΙμΜ corresponds to 0.5- 2^g/ml.

61

142690 V12/DC

62

142690 V12/DC

Example 3

MRSA challenge. We used THAM trisguanide in a preliminary animal study, using a mouse model of systemic MRSA infection (Watkins et al, (2011) PLoS One. 6:el9939). Groups of three mice were challenged with a lethal dose of MRSA (US A300 strain, 5 x 10⁷ CFU given i.p.) and treated as follows: 1) the control group was infected, but untreated, 2) the second group was injected i.p. with 0.1 mL of 100 μΜ THAM trisphenylguanide

immediately after infection, and 3) the third group was injected i.p. with 0.1 mL of 100 μΜ THAM trisphenylguanide one hour post infection. At eight hours post infection, the mice were euthanized. An intraperitoneal wash using 10 ml PBS was preformed to recover bacteria within the peritoneal cavity. The heart and kidney were removed for evaluation of systemic infection. Organs were macerated, vortexed and plated at multiple dilutions on tryptic soy agar (TSA) plates. The TSA plates were incubated overnight at 37°C and counted the next day.

In mice that were given an i.p. injection of 0.1 mL 100 μΜ THAM trisguanide an hour later, this treatment substantially reduced morbidity and decreased bacterial CFU counts in their kidneys by 1-2 logs, compared to mock-treated controls, when the mice were sacrificed 8 hours post infection (Figure 5). The compound with the widest spectrum of activity, THAM trisphenylguanide (THAM-30G), has also been used for preliminary tests in mice. In a dose-escalation toxicity trial, groups of three mice were injected i.p. with 0.1 mL of 0, 10, 50, and 100 μΜ THAM trisphenylguanide. After 2 hours, no effects were seen in a standard panel of serum chemistry and enzyme levels. We then gave the 100 μΜ dose to two groups of 8 mice, either at the same time as, or one hour after, an i.p. challenge with a lethal dose of MRSA. The mice treated right after the challenge averaged a 3-log reduction in CFU in kidney tissue after 8 hours, compared to untreated mice. Mice treated an hour after challenge had a 1-log reduction.

No abnormalities in serum chemistry panels were observed at any of the doses of either compound. The compounds were not overtly cytotoxic in vivo, since no hemolysis was observed in the blood from treated mice, and no plasma membrane damage in human PBMCs and PMNs was observed by propidium iodide staining and flow cytometry.

Example 4

CXCR4-T140 cross-link inhibition assay. To determine anti-HIV activity, the compounds were tested for CXCR4 binding by inhibiting the cross-linking of a photoactive, fluorescent derivative of the known CXCR4 binding peptide T140. Serial dilutions of the inhibitor compounds were incubated with approximately 1.5 ^x 10⁵ CXCR4 expressing cells (syn-CXCR4-C9 in Cf2Th) (Babcock et al, (2001) J Biol Chem 276:38433-40) for 30 minutes on ice. The photoactive, fluorescent T140 peptide was added to a final concentration of 75 nM, and incubated on ice for an additional 30 minutes. Cross-linking of the T140 peptide to the receptor was carried out by irradiation at 365 nm in a Rayonet Photochemical Reaction chamber (Southern New England Ultraviolet Company, Hamden, CT) 4 times for 5 minutes with 5 minutes of cooling on ice between irradiations. The cell pellets were spun down, washed with 1 x PBS, and resuspended in PAGE sample buffer with 40 mM

dithiothreitol and run on an SDS-PAGE gel. The gels were imaged on a Bio-Rad Molecular Imager FX (Hercules, CA) and the fluorescent CXCR4 bands were quantified with the Bio- Rad Quantity One software. A representative gel and analysis are shown in Figure 6 for the dendrimer derivative DNT2200 octaguanide. The concentration of inhibitor is marked at the top of each lane. The intensity of the fluorescent CXCR4 bands (doublet indicated by arrow) were quantified and plotted with Sigma Plot to determine an approximate IC₅₀ value (~ 1 μΜ). A Western blot of the gel incubated with MAb 1D4 was used to confirm the identity of the CXCR4 bands and their uniformity across the gel (not shown). The amount of fluorescence was quantified as the fluorescence density of a box encompassing the doublet band (CXCR4) in the middle of the gel.

The results of the CXCR4-T140 cross-link inhibition assay are shown in Table 4. IC₅₀ values are given for the starting material amines and the respective guanide, biguanide, and phenylguanide derivatives. Most of the starting amines had IC₅₀ values greater than or equal to the highest concentration tested (200 μΜ), with the exception of spermine, which inhibited the T140 cross-linking with an IC₅₀ of 75 μΜ.

As a general rule, the inhibition activity increased in the order amines < biguanides < guanides < phenylguanides. However, there are a couple of notable exceptions. The butanediamine was only active as the guanide derivative, with an IC₅₀ of 50 μΜ. For trishexylaminomelamine (THAM), the guanide and biguanide derivatives were active, but interestingly, the phenylguanide compound showed no inhibition. The derivatives of linear polyamines (butanediamine, hexanediamine, diethylenetriamine, spermidine, and spermine) generally increased in activity as they increased in size. As a group, the spermidine and spermine derivatives were the most active, and the most inhibition was exhibited by spermidine bisphenylguanide and spermidine trisphenylguanide with IC₅₀ values of 200 nM. The melamine core guanide derivatives (TEAM, TBAM, THAM) increased in activity as the chain length increased (ethyl < buty < hexyl). Conversely, the corresponding phenylguanides showed the opposite trend, with the THAM bisphenylguanide actually showing no inhibition at the highest concentration tested (200 mM). The dendrimer derivatives (PAMAM-G0, DNT2300, DNT2200) were generally equally active as the number of reactive amines varied from 4 to 6 to 8.

Example 5

Inhibition of HIV infection. Six of the most active compounds, and the amines from which they were derived, were selected for preliminary testing as HIV infection inhibitors. Spermidine, spermine, DNT2300, and their guanide and phenylguanide derivatives were tested for their ability to inhibit HIV infection at 10μΜ in the TZM-bl assay. The compounds were screened against CXCR4, CCR5, and R5/X4 tropic virus strains. The spermidine and spermine phenylguanides were effective against both X4 strains, with no inhibition of the R5/X4 strain and limited inhibition of the R5 strain (see Figure 7). The series (amine, guanide, and phenylguanide) of the most active derivatives (spermidine, spermine, and DNT2300) in the T140 cross-link inhibition assay were screened for anti-HIV activity. Cell- free virus supematants were prepared in PHA blast cultures as described elsewhere

(Krowicka et al, (2008) AIDS Res Hum Retroviruses 24:957-67). TZM-bl cells (Wei et al, (2002) Antimicrob Agents Chemother 46: 1896-905) were obtained from the NIH AIDS Research and Reference Reagent Program; 4 ^χ 10⁴ cells were plated per well. CXCR4 inhibitors were added to the cells for 1 hour at 37°. Predetermined dilutions of the virus supematants in 15 μg/ml DEAE-dextran (Sigma) were then added to the wells containing the cells plus inhibitor. TZM-bl cells were plated and pre-incubated with the inhibitors at a final concentration of 10 μΜ. Previously titered viral supematants for HIV clones NL4-3 (Figure 7A), 92HT599 (Figure 7B), 92HT594 (Figure 7C), and Ba-L (Figure 7D) were added and the cells incubated for 3 days. Cells were lysed and the TAT-driven reporter luciferase activity measured with the Bright-Glo Luciferase assay (Bright-Glo Luciferase assay, Promega, Madison WI).

The underivatized amine spermine appeared to be active against both R5 and X4 strains at this concentration. However, in a second TZM-bl assay ran to investigate the dose- response of the active phenylguanides and their respective amine starting materials, the phenylguanides were active at approximately 10-fold lower concentrations than the corresponding amines against three X4 strains (Figures 8 and 9A-9C). Increased activity against CXCR4-using viral strains were observed with the phenylguanides compared to the underivatized parent amines. None of the compounds showed significant activity (>2-fold reduction) against an R5 vims. At 10 μΜ, spermine did not significantly inhibit fluorescent T-140 binding to CXCR4 (see above) or binding of a fluorescent maraviroc analog to CCR5, nor was it cytotoxic to TZM-bl cells (data not shown). To perform the CCR5-maraviroc analog cross-link inhibition assay, a maraviroc analog containing a benzophenone cross- linking group and a fluorescein tag were prepared. This compound was used to test several compounds of the present invention for interaction with CCR5 in an assay similar to the CXCR4-T140 inhibition assay. Example 6

Cytotoxicity. Compound cytotoxicity was evaluated using the CellTiter 96®

Aqueous Non-Radioactive Cell Proliferation Assay (MTS) following the manufacturer's instructions (Promega, Madison, WI) using both the human breast cancer cell line MDA-MB- 231 and HaCaT human keratinocytes (Boukamp et al, (1988) J Cell Biol. 106:761-71). The HaCaT cell line was grown in cell culture medium containing human keratinocyte growth factor. Cells were harvested using Tryspin/EDTA and counted for 1 x 10⁵ cells per well in a 96-well plate. Ten concentrations covering five orders of magnitude were tested with six replicates per concentration. Cells were incubated for 48-72 hours at 37°C with 5% C0₂. MTS/PMS solution was added to the wells and absorbance was measured at 490 nm 2-4 h later. The same protocol was used for the MDA-MB-231 cells. The toxicity of the compounds in a CXCR4 expressing human breast cancer cell line (MDA-MB-231) for all of the compounds is shown in Table 5. Toxicity results in TZM-bl cells for spermidine phenylguanide and the spermidine control are shown in Figure 9D. The majority of the compounds showed no toxicity at the highest concentrations tested against the MDA-MB-231 cells. Spermidine and spermidine phenylguanide both had CC₅₀ values of approximately 1000 μΜ against the TZM-bl cells while showing HIV inhibition at <5 μΜ (Figures 9A-9C).

Table 5. Compound cytotoxicity against CXCR4 expressing human breast carcinoma MB-231) cells and human keratinocytes (HaCat) cells.

Example 7

Most of the mortality associated with breast cancer is due to the metastatic spread of tumor cells to sites distant from the primary locus and the establishment of secondary tumors. Adjuvant chemotherapy is commonly given to prevent the growth of these secondary tumors, but is associated with unpleasant side-effects because it also affects normal proliferating cells. Endocrine therapies may have fewer side-effects, but secondary tumors may lose the estrogen-dependence of the primary tumor, and thereby become resistant to this type of therapy. This experiment tested the hypothesis that adjuvant chemotherapy should target the mechanisms by which cells leave the primary tumor and travel to distal sites. Drugs that inhibit these processes need not be cytotoxic and could be given for prolonged periods after detection of a primary breast tumor, or even prophylactically to those at high risk for developing breast cancer.

CXCR4, a member of the chemokine receptor family of G-protein-coupled receptors (GPCRs) has been shown to have a major role in metastasis. CXCR4 is involved in chemotaxis, cell proliferation, and angiogenesis when bound by its ligand SDF-1. It is found on a wide range of tumor cells and high levels are associated with higher grade and poor prognosis of breast and other cancers. Blocking the interaction between CXCR4 and its only ligand SDF-1 dramatically reduces breast and other cancer metastases in mouse models. Several compounds that antagonize CXCR4 are being developed as potential anti-tumor drugs, and some have gone into clinical trials. Many of these have shown toxic side-effects in animal studies and/or poor bioavailability. We have synthesized a series of molecules containing guanidine or biguanide functional groups that are CXCR4 antagonists. We have shown that all of these compounds have very low in vitro cytotoxicity (see Example 5). Preliminary "wound healing" assays with human breast cancer cells in culture showed that some of these compounds inhibit cell migration at concentrations 10- to 100-fold lower than their CC50.

To perform the wound healing assay, MDA-MB-231 cells are grown to a confluent layer in small Petri dishes in DMEM/F12 media. Once the cells are confluent, the media is briefly removed and a 200 ul pipette tip is used to carve a straight line into the cells using a ruler for guidance. Media is re-added to the cells +/- drug. Initial measurements are taken on the width of the wound. Measurements are taken for the next 2-3 days for two to three times a day at about the same location on the wound. In preliminary experiments, 10 μΜ spermine bisguanide inhibited the growth and migration of the cells to close the "wound" by a factor of ~2 compared to a no-drug control, which was comparable to the result obtained with 10 nM T-140.

Example 8

These compounds of the present invention may be used as effective inhibitors of breast cancer metastasis in vivo, and we will test this in a mouse model with a lung colonization assay: three test inhibitor doses will be tested in CB/17 SCID mice. Each mouse will be injected intravenously via the tail vein with 50,000 monodispersed MDA-MB-231 tumor cells in 0.2 ml saline. The test inhibitor will be given intravenously twice (a maximal volume of 0.2 ml) weekly starting 2 hours after the tumor injection. After 3 weeks, the mice will be sacrificed and the metastatic load enumerated. Blood samples will be taken at this time by cardiac puncture and clinical chemistry will be run on the sera to establish if any unexpected toxicity occurred. After checking for any extrapulmonary tumor colonies, the lung sets are retrieved and placed in Bouin's fixative. After 3 days, the individual lung lobes are examined under a dissecting microscope, and all surface tumor colonies are counted. For the lung colony assay, we will use 8-10 mice per group to establish statistically significant results. For one inhibitor/tumor combination we will have 3 test groups (3 doses) and one control group. We expect to see a dose-dependent reduction in the number of tumor colonies in the mice treated with our compounds of the present invention. Example 9

Starting material amine compounds. For this example and those following, putrescine dihydrochloride and spermine tetrahydrochloride were purchased from Sigma (St. Louis, MO). 1 ,4-diaminobutane, 1,6-hexanediamine dihydrochloride, 1,6-hexanediamine, diethylenetriamine, and spermidine were purchased from Acros (Morris Plains, NJ).

Starburst®PAMAM G(0.0) was purchased from Dendritech Inc. (Midland, MI). Priostar™ dendrimers DNT-2200 and DNT-2300 were purchased from Dendritic Nanotechnologies, Inc. (Mt. Pleasant, MI).

Trishexylaminomelamine (THAM) synthesis. Trishexylaminomelamine was synthesized following the method of Kaiser et al. (1951) Journal of the American Chemical Society 73:2984-2986. Cyanuric chloride (TCI America, Tokyo) was reacted with a 3.3 molar ratio of N-BOC-l,6-diaminohexane (Alfa Aesar, Ward Hill, MA) in refluxing water for 1.25 hours. The pH of the solution was monitored with phenolphthalein and the pink color maintained by gradual addition of a 0.4M sodium carbonate solution. The reaction was lyophilized and the BOC groups removed by treating the residue with 100% trifluoroacetic acid (TFA) for 2h at room temperature (rt). The deprotected product was purified by reverse- phase HPLC.

Example 10

Trisethylaminomelamine (TEAM) synthesis. The synthesis was begun as above with N-BOC-l,2-diaminoethane produced as previously described (Muller et al, (1997)

Journal of Organic Chemistry 62:411-416). The initial reaction gave the bis addition product (after deprotection) with one unreacted chlorine. Treatment of this compound with a concentrated ammonia solution in water resulted in the formation of a dimeric compound (BEMA dimer). Treatment of the bis addition product before deprotection with excess ethylenediamine, followed by deprotection, gave the desired trisethylamino product which was purified by reverse-phase HPLC.

Example 11

Trisbutylaminomelamine (TBAM) synthesis. The synthesis was similar to the

TEAM synthesis. The initial bis addition product, after deprotection, was treated with excess 1 ,4-butanediamine and refluxed for 2h in water. Purification by HPLC under acidic conditions did not result in separation of the desired product from the excess diamine.

However, under basic conditions the product bound and was not eluted. Subsequent washing with 0.1% TFA in water eluted the desired product without the contaminating diamine.

Example 12

Trisoctylaminomelamine (TOAM) synthesis. N-BOC-l,8-diaminooctane was prepared as previously described (Dardonville et al. Bioorg. Med. Chem. (2006), 14:6570, and purified by silica chromatography (2: 1 CHC13:CH30H). The synthesis of

trisoctylaminomelamine (TOAM) was similar to the THAM synthesis above. The only exception was that after initial reaction of the BOC protected diamine with cyanuric chloride in refluxing water for 2hours, an equal volume of acetonitrile was added and the reaction continued, at reflux, for an additional hour.

Example 13

Synthesis of guanide derivatives. The reaction of polyamine starting materials was carried out using 10% excess O-methylisourea sulfate salt (MP Biomedicals) in water at basic pH. Free amine compounds were added to the O-methylisourea in water and incubated at 37- 42°C for 1-2 hours. Reactions with acidified amine starting materials (HC1 or TFA salts) were neutralized with triethylamine (TEA) before being allowed to react. Products were purified by reverse phase HPLC and characterized by MALDI mass spectrometry.

Example 14

Synthesis of biguanide derivatives. S-methyl-guanylisothiouronium iodide was prepared as previously described (Eilingsfeld et al, (1967) Chemische Berichte-Recueil 100: 1874-91). Briefly, iodomethane (Acros) was added dropwise to a suspension of 2-imino- 4-thiobiuret (Aldrich) in 100% ethanol while stirring at rt. The reaction flask was shaken at 37°C for 2 hours. The solvent was removed by evaporation and the residue was washed with cold diethyl ether to give a white, crystalline solid which was collected by filtration and used without further purification. Addition of the biguanide reagent (S-methyl- guanylisothiouronium iodide, in excess) to the amine compounds was done in 100% EtOH as previously described (38) at rt or 60°C, or in water at rt, 37°C, or 65°C. The pH of the reactions were monitored and adjusted with TEA or sodium carbonate when it fell below pH~8. Reaction times varied between 5h and five days. Products were purified and characterized as above.

Example 15

Synthesis of phenylguanide derivatives. S-methyl-N-phenylisothiouronium iodide was prepared as above by addition of methyl iodide to l-phenyl-2-thiourea (Acros).

Addition to the amine compounds was accomplished by refluxing 10% excess of the S- methylthiourea compound with the amine in 50% acetonitrile/water overnight. Reactions with acidified amine starting materials (HCl or TFA salts) were neutralized with

triethylamine (TEA) as above. Products were purified and characterized as above.

Example 16

Synthesis of 1-naphthylguanide derivatives. S-methyl-N-l-naphthylisothiouronium iodide was prepared as above by addition of methyl iodide to N-(l-naphthyl) thiourea (Acros). Addition to the amine compounds was accomplished by refluxing 10%> excess of the S-methylthiourea compound with the amine in 50% acetonitrile/water overnight.

Reactions with acidified amine starting materials (HCl or TFA salts) were neutralized with triethylamine (TEA) as above. Products were purified and characterized as above. Example 17

Synthesis of 2-naphthylguanide derivatives. N-(2-naphthyl) thiourea was prepared as previously described (Nair, J. Ind. Chem. Soc. (1963) 10:953) from 2-aminonaphthylene (Toronto Research Chemicals). The preparation of S-methyl-N-2-naphthylisothiouronium iodide and the subsequent reaction with amine compounds was performed as above for 1- naphthylguanide.

Example 18

Synthesis of a photoactive, fluorescent T140 peptide. A fluorescent, photoactive cross-linkable derivative of the CXCR4 binding peptide T140 (Ac-RRNalCYRBpaD- KPYRCitCR, Nal=naphthylalanine, Cit=citruline, Bpa=4-benzoylphenylalanine) (Tamamura et al., (2003) Org Biomol Chem 1 :3656-62; Tamamura et al., (1998) Biochem Biophys Res Commun 253:877-82) was synthesized on a solid support using standard Fmoc chemistry. Fmoc amino acids with appropriate side chain protecting groups were purchased from Advanced Chemtech (Louisville, KY). After cleavage from the resin and side chain deprotection, the crude peptide was purified by reverse-phase HPLC. A dilute solution of the peptide in 10 mM ammonium bicarbonate, pH=8, was stirred while bubbling air through the solution at 4°C until oxidation of the intrapeptide disulfide bond was complete as analyzed by MALDI mass spectrometry. The solution was lyophilized and the oxidized peptide purified by reverse phase HPLC .

The purified peptide was fluorescently labeled on the ε-amine of the D-lysine residue by reaction with N-hydroxysuccinimide activated 6-(fluorescein-5-(and-6)- carboxamido)hexanoic acid (Invitrogen, Carlsbad, CA) in dry DMF with 10% pyridine overnight at 4°C. The labeled peptide was purified by reverse phase HPLC.

Example 19

Various compounds were synthesized as described in the preceding examples and then characterized by Matrix- Assisted Laser Desorption/lonization (MALDI) Time of Flight (TOF) mass spectrometry and by Nuclear Magnetic Resonance (NMR) spectroscopy using standard techniques (see Table 6).

Table 6. Compound characterization data.

Starting amines:

Trisethylaminomelamine (TEAM):

MALDI: 256.7 (expected 256.2 MH⁺); ¹H-NMR (D₂0): 3.05 (bs, 1H), 3.55 (bs, 1H), 8.25 (NH - exchangeable).

Bisethylaminomelamine dimer (BEMA dimer):

MALDI: 392.0 (expected 391.3 MH⁺).

Trisbutylaminomelamine (TBAM):

MALDI: 341.0 (expected 340.3 MH⁺); ¾-NMR (D₂0): 1.6 (bs, 1H), 2.9 (bs, 1H).

Trishexylaminomelamine (THAM):

MALDI: 424.3 (expected 424.4 MH⁺); ¾-NMR (D₂0): 1.15 (bs, 4H), 1.4 (t, 2H), 1.5 (t, 2H), 2.8 (t, 2H), 3.15 (t, 1H), 3.25 (t, 1H).

Trisoctylaminomelamine (TO AM):

MALDI: 508.3 (expected 508.5 MH⁺).

Derivatives:

Butanediamine bisguanide:

MALDI: 173.6 (expected 173.2 MET).

Butanediamine bisbiguanide:

MALDI: 257.9 (expected 257.2 MH⁺).

Butanediamine bisphenylguanide:

MALDI: 325.6 (expected 325.2 MH⁺).

Hexanediamine bisguanide:

MALDI: 201.6 (expected 201.2 MH⁺).

Hexanediamine monophenylguanide:

MALDI: 235.8 (expected 235.2 MH⁺).

Hexanediamine bisphenylguanide:

MALDI: 353.8 (expected 353.2 MH⁺).

Diethylenetriamine (DETA) bisguanide:

MALDI: 188.5 (expected 188.2 MH⁺).

Diethylenetriamine (DETA) bisbiguanide:

MALDI: 272.9 (expected 272.2 MH ). Diethylenetriamine (DETA) bisphenylguanide:

MALDI: 340.7 (expected 340.2 MH ).

Spermidine trisguanide:

MALDI: 272.8 (expected 272.2 MH⁺); ¹H-NMR (D₂0): 1.5 (m, 1H), 1.6 (m, 1H), 1.85 (m,

1H), 2.9-3.0 (t, t, 2H), 3.1 (t, 1H), 3.25 (t, 1H).

Spermidine biguanides (mixture of mono- and bisbiguanide):

MALDI: 230.6, & 314.6 (expected 229.2, & 314.3 MH⁺); 1H-NMR (D₂0): 1.55 (bs, 4H), 1.9

(bs, 2H), 2.9 (bs, 4H), 3.25 (bs, 2H), 3.3 (bs, 2H), 7.4-7.7 (m, 8H), 7.8-8.0 (m, 6H).

Spermidine bisbiguanide:

MALDI: 382.9 (expected 382.3 MH⁺).

Spermidine trisbiguanide:

MALDI: 501.0 (expected 500.3 MH⁺).

Spermidine trisphenylguanide:

MALDI: 500.8 (expected 500.3 MH⁺).

Spermidine bis-l-naphthylguanide:

MALDI: 482.2 (expected 482.3 MH⁺); ¾-NMR (D₂0): 1.6 (bs), 1.9 (m), 2.85 (m), 2.9-3.0 (m).

Spermidine bis-2-naphthylguanide:

MALDI: 482.2 (expected 482.3 MH⁺); ¾-NMR (D₂0/CD₃-CN): 2.05 (bs, 4H), 2.4 (under CD₃-CN peak), 3.4 (bs, 4H), 3.7 (bs, 2H), 3.75 (bs, 2H), 7.7-7.85 (d, 2H), 8.0 (bs, 4H), 8.2 (s, 2H), 8.3-8.5 (m, 6H).

Spermine bisguanide:

MALDI: 287 (expected 287.3 MH⁺); 1H-NMR (D₂0): 1.65 (bs, 1H), 1.85 (bm, 1H), 2.95 (m, 2H), 3.15 (t, 1H).

Spermine bisbiguanide:

MALDI: 371.8 (expected 371.3 MH⁺); ¾-NMR (D₂0): 1.6 (bs, 1H), 1.9 (m, 1H), 2.95 (m, 2H), 3.25 (bs, 1H).

Spermine tetraphenylguanide:

MALDI: 675.8 (expected 675.4 MH⁺); ¾-NMR (D₂0): 1.85 (bs, 1H), 2.15 (bs, 1H), 3.15 (bs, 1H), 3.45 (m, 1H), 3.65 (bs, 1H), 7.35-7.45 (m, 2H), 7.45-7.65 (m, 2H), 7.8-7.95 (m, 1H).

Trisethylaminomelamine (TEAM) trisguanide: MALDI: 381.5 (expected 382.3 MH⁺); 1H-NMR (D₂0): 3.25 (bs, 2H), 3.4 (t, IH), 3.5 (bs, IH).

Trisethylaminomelamine (TEAM) monobiguanide:

MALDI: 340.8 (expected 340.2 MH⁺).

Trisethylaminomelamine (TEAM) trisphenylguanide:

MALDI: 609.9 (expected 610.4 MH⁺); ¾-NMR (D₂0): 3.3 (bs, 2H), 3.4 (bs, 2H), 7.0 (bs, 2H), 7.2-7.35 (m, 3H).

Bisethylaminomelamine dimer (BEMA dimer) bisguanide:

MALDI: 476.0 (expected 475.3 MH⁺).

Bisethylaminomelamine dimer (BEMA dimer) bisphenylguanide:

MALDI: 627.9 (expected 627.4 MH⁺).

Trisbutylaminomelamine (TBAM) trisguanide:

MALDI: 467.1 (expected 466.4 MH⁺).

Trisbutylaminomelamine (TBAM) bisphenylguanide:

MALDI: 577.2 (expected 576.4 MH⁺).

Trisbutylaminomelamine (TBAM) trisphenylguanide:

MALDI: 697.0 (expected 694.5 MH⁺).

Trisbutylaminomelamine (TBAM) monobiguanide/ bisbiguanide mixture:

MALDI: 424.8 (expected 424.3 MH⁺) and MALDI: 508.9 (expected 508.4 MH⁺).

Trishexylaminomelamine (THAM) trisguanide:

MALDI: 550.1 (expected 550.5 MH⁺); ^XH-NMR (D₂0/CD₃-CN): 1.2 (bs, 4H), 1.45 (bs, 4H),

3.0 (bs, 2H), 3.15 (t, IH), 3.25 (t, IH); 1H-NMR (D₂0): 1.15 (bs, 4H), 1.45 (bs, 4H), 3.0 (t, 2H), 3.2 (t, IH), 3.3 (t, IH).

Trishexylaminomelamine (THAM) bisbiguanide:

MALDI: 592.0 (expected 592.5 MH⁺); ¾-NMR (D₂0/CD₃-CN): 1.2 (bs, 6H), 1.45 (bs, 6H),

3.1 (bs, 2H), 3.2 (bs, 2H), 3.3 (bs, 2H).

Trishexylaminomelamine (THAM) trisphenylguanide:

MALDI: 777.7 (expected 778.5 MH⁺); ¾-NMR (D₂0/CD₃-CN): 1.5 (bs, 4H), 1.65 (bs, 4H), 3.4 (bs, 3H), 3.5 (bs, IH), 7.3 (bs, 2H), 7.4 (t, IH), 7.5 (t, 2H).

Trisoctylaminomelamine (TOAM) trisguanide:

MALDI: 634.4 (expected 634.5 MH⁺); ^XH-NMR (D₂0/CD₃-CN): 1.65 (bs, 12H), 1.9 (bs, 6H), 3.45 (bs, 3H), 3.65 (bs, IH), 3.75 (bs, 2H).

Trisoctylaminomelamine (TOAM) bisphenylguanide: MALDI: 744.5 (expected 744.6 MH⁺); 1H-NMR (D₂0/CD₃-CN): 1.7 (bs, 24H), 1.95 (bs, 12H), 3.25 (m, 2H) 3.55 (bs, 4H), 3.65 (bs, 2H), 3.75 (bs, 4H), 7.65 (d, 4H), 7.8 (t, 2H), 7.9 (t, 4H).

Trisoctylaminomelamine (TO AM) trisphenylguanide:

MALDI: 862.3 (expected 862.6 MH⁺); 1H-NMR (D₂0/CD₃-CN): 1.7 (bs, 4H), 1.95 (bs, 2H), 3.6 (bs, 4H), 3.7 (bs, 2H), 3.8 (bs, 2H), 7.65 (bs, 2H), 7.8 (t, 1H), 7.9 (t, 2H).

PAMAM-GO tetraguanide:

MALDI: 686.3 (expected 685.5 MH⁺); 1H-NMR (D₂0): 2.5 (bs), 3.0 (bs), 3.2 (m), 3.25 (m). PAMAM-GO biguanides (mixture of bis-, tri-, and tetrabiguanide):

MALDI: 686.2, 770.3, & 854.4 (expected 685.5, 769.5, & 853.6 MH⁺); 1H-NMR (D₂0): 2.4 (m, 2H), 2.65 (bs, 1H), 2.8 (bs, 2H), 3.0 (t, 1H), 3.2 (bs, 2H), 3.4 (t, 1H).

DNT2300 hexaguanide:

MALDI: 864.4 (expected 864.6 MH⁺); ¾-NMR (D₂0): 0.7 (t, 3H), 1.2 (m, 2H), 3.2-3.35 (m, 12H), 3.35-3.55 (m, 18H), 3.55-3.7 (bs, 12H), 4.15 (bs, 3H).

DNT2300 biguanides (mixture of terra-, penta-, and hexabiguanide):

MALDI: 942.9, 1026.9, & 1111.0 (expected 948.7, 1032.7, & 1116.8 MH⁺); ¹H-NMR (D₂0):

0.7 (t), 1.2 (q), 3.2-3.7 (m), 4.2 (bs).

DNT2300 hexaphenylguanide:

MALDI: 1322.1 (¹ C isomer) (expected 1320.8 MH⁺).

DNT2200 octaguanide:

MALDI: 1109.4 (expected 1109.8 MH⁺); ¹H-NMR (D₂0): 2.55 (bs, 2H), 2.65 (bs, 2H, 2.75

(bs, 2H), 3.2 (bs, 4H), 3.25-3.45 (m, 4H), 3.9 (bs, 1H).

DNT2200 biguanides (mixture of bis-, tri-, tetra, and pentabiguanide):

MALDI: 942.2, 1026.3, 1108.5, & 1194.6 (expected 941.7, 1025.8, 1109.8, & 1193.8 MH⁺). DNT2200 octaphenylguanide:

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Claims

CLAIMS:

1. A compound having the following structure:

or a pharmaceutically-acceptable salt or ester thereof, wherein:

Ri is selected from the group consisting of:

hydrogen and

R₂ is selected from the group consisting of:

and

wherein M is selected from the group consisting of phenyl, napthalene, and a monocyclic or bicyclic heteroaromatic group, wherein phenyl, napthalene, and the monocyclic or bicyclic heteroaromatic group are each optionally substituted with one or more substituents independently selected from the group consisting of halo, hydroxy, alkyl, linear or branched Ci-C₆ alkyl, linear or branched C₂-C₆ alkenyl, linear or branched C₂-C₆ alkynyl, cycloalkyl, haloalkyl, hydroxyalkyl, alkoxy and alkoxyalkyl; with the proviso that at least one R must be one of structures I-III.

2. A compound of claim 1, wherein each instance of R is independently selected from the group consisting of: -NH₂, structures I - II, and the following structure:

3. A compound of claim 1, selected from the group consisting of:

90

and pharmaceutically acceptable salts or esters thereof.

4. The compound of claim 3, wherein each instance of R is independently selected from the group consisting of: -NH₂, structures I-II, and the following structure:

5. A compound having the following structure:

or a pharmaceutically-acceptable salt or ester thereof, wherein:

Z is selected from the group consisting of straight chained or branched C1-C12 alkyl, straight chained or branched C2-C12 alkenyl, straight chained or branched C2-C12 alkynyl; wherein said straight chained or branched alkyl, alkenyl or alkynyls may include one or more spacer moieties selected from the group consisting of O, S, NH(C=0), (C=0)NH, 0(C=0),

(C=0)0, (C=0) and

IA.

IIA.

IIIA.

wherein M is selected from the group consisting of phenyl, napthalene, and a monocyclic or bicyclic heteroaromatic group, wherein phenyl, napthalene, and the monocyclic or bicyclic heteroaromatic group are each optionally substituted with one or more substituents independently selected from the group consisting of halo, hydroxy, linear or branched Ci-C₆ alkyl, linear or branched C₂-C₆ alkenyl, linear or branched C₂-C₆ alkynyl, cycloalkyl, haloalkyl, hydroxyalkyl, alkoxy and alkoxyalkyl; and each instance of R is independently selected from the group consisting of: hydrogen, -CH₃, -NH_2,

III.

wherein M is selected from the group consisting of phenyl, napthalene, and a monocyclic or bicyclic heteroaromatic group, wherein phenyl, napthalene, and the monocyclic or bicyclic heteroaromatic group are each optionally substituted with one or more substituents independently selected from the group consisting of halo, hydroxy, linear or branched Ci-C₆ alkyl, linear or branched C₂-C₆ alkenyl, linear or branched C₂-C₆ alkynyl, cycloalkyl, haloalkyl, hydroxyalkyl, alkoxy and alkoxyalkyl

and

6. The compound of claim 5, wherein each instance of R is independently selected from the group consisting of -NH₂, structures I - II, and the following structure;

each instance of Y is independently selected from the group consisting of hydrog structures IA - IIA and the following structure:

7. A compound of claim 5, selected from the group consisting of

harmaceutically acceptable salts or esters thereof.

8. The compound of claim 7, wherein each instance of R is independently selected from the group consisting of -NH₂, structures I - II and the following structure:

and each instance of Y is independently selected from the group consisting of hydrogen, structures IA - II A, and the following structure:

9. A compound of claim 7, selected from the group consisting of

10. The compound of claim 9, wherein R and Y comprise 3 or more structures independently selected from the group consisting of:

1 1. A composition comprising:

a compound having the following structure:

or a pharmaceutically-acceptable salt or ester thereof, wherein:

Ri is selected from the group consisting of: hydrogen and

R₂ is selected from the group consisting of:

wherein M is selected from the group consisting of phenyl, napthalene, and a monocyclic or bicyclic heteroaromatic group, wherein phenyl, napthalene, and the monocyclic or bicyclic heteroaromatic group are each optionally substituted with one or more substituents independently selected from the group consisting of halo, hydroxy, linear or branched Ci-C₆ alkyl, linear or branched C₂-C₆ alkenyl, linear or branched C₂-C₆ alkynyl, cycloalkyl, haloalkyl, hydroxyalkyl, alkoxy and alkoxyalkyl; and one or more pharmaceutically acceptable excipients.

12. A composition comprising a compound of claim 4 and one or more pharmaceutically acceptable excipients.

13. The composition of claim 11, wherein said compound binds to a G-protein coupled receptor.

14. The composition of claim 13, wherein said G-protein coupled receptor is selected from one or more of the group consisting of CCRl, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCRIO, CXCRl, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CXCR7, XCR1 and CX3CR1.

15. The composition of claim 14, wherein said G-protein coupled receptor acts as a co- receptor for HIV- 1 infection.

16. The composition of claim 15, wherein said compound binds CXCR4 with an IC50 less than about 200 11M.

17. The composition of claim 16, wherein said compound binds CXCR4 with an IC50 less than about 200 μΜ in competition cross-link inhibition assays with T-140.

18. The composition of claim 17, wherein said IC50 is less than about 10 μΜ.

19. The composition of claim 17, wherein said IC50 is less than about 2.5 μΜ.

20. A composition comprising a compound having the following structure:

wherein:

Z is selected from the group consisting of straight chained or branched C1-C12 alkyl, straight chained or branched C2-C12 alkenyl, straight chained or branched C2-C12 alkynyl; wherein said straight chained or branched alkyl, alkenyl or alkynyls may include one or more spacer moieties selected from the group consisting of O, S, NH(C=0), (C=0)NH, 0(C=0), (C=0)0, (C=0) and

IA.

wherein M is selected from the group consisting of phenyl, napthalene, and a monocyclic or bicyclic heteroaromatic group, wherein phenyl, napthalene, and the monocyclic or bicyclic heteroaromatic group are each optionally substituted with one or more substituents independently selected from the group consisting of halo, hydroxy, linear or branched Ci-C₆ alkyl, linear or branched C₂-C₆ alkenyl, linear or branched C₂-C₆ alkynyl, cycloalkyl, haloalkyl, hydroxyalkyl, alkoxy and alkoxyalkyl; and each instance of R is independently selected from the group consisting of: hydrogen, -CH₃, -NH₂,

III.

wherein M is selected from the group consisting of phenyl, napthalene, and a monocyclic or bicyclic heteroaromatic group, wherein phenyl, napthalene, and the monocyclic or bicyclic heteroaromatic group are each optionally substituted with one or more substituents independently selected from the group consisting of halo, hydroxy, linear or branched Ci-C₆ alkyl, linear or branched C₂-C₆ alkenyl, linear or branched C₂-C₆ alkynyl, cycloalkyl, haloalkyl, hydroxyalkyl, alkoxy and alkoxyalkyl; and

and one or more pharmaceutically acceptable excipients.

21. A composition comprising the compound of claim 8 and one or more pharmaceutically acceptable excipients.

22. The composition of claim 20, wherein said compound binds to a G-protein coupled receptor.

23. The composition of claim 22, wherein said G-protein coupled receptor is selected from one or more of the group consisting of CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CXCR7, XCR1 and CX3CRl .

24. The composition of claim 23, wherein said G-protein coupled receptor acts as a co- receptor for HIV- 1 infection..

25. The composition of claim 24, wherein said compound binds CXCR4 with an IC₅₀ less than about 200 nM.

26. The composition of claim 25, wherein said compound binds CXCR4 with an IC₅₀ less than about 200 μΜ in competition cross-link inhibition assays with T-140.

27. The composition of claim 26, wherein said IC₅₀ is less than about 10 μΜ.

28. The composition of claim 26, wherein aid IC₅₀ is less than about 2.5 μΜ.

29. A method comprising administering an effective amount of the composition of claim 11 or claim 20 to a patient.

30. The method of claim 29, wherein said patient is in need of said composition , as a cytokine antagonist; to improve skin rejuvenation; to increase stem-cell mobilization; to increase hematopoiesis; to improve wound healing; to treat, inhibit and/or prevent HIV; to treat, inhibit and/or prevent cancer; to treat, inhibit and/or prevent a bacterial infection; and to treat, inhibit and/or prevent a viral infection.

31. The method of claim 30, wherein said patient is suffering from an HIV infection.

32. The method of claim 31 , wherein said HIV is a strain selected from one or more of the group consisting of R5 and X4 from HIV-1.

33. The method of claim 30, wherein said patient is suffering from a bacterial infection.

34. The method of claim 33, wherein said bacterial infection is an infection of Acinetobacter baumannii, Burkholderia Cepacia, Enterococcus faecalis, Escherichia coli, Pseudomonas aeruginosa or Staphylococcus aureus.

35. The method of claim 34, wherein said bacterial infection is an infection of methicillin- resistant Staphylococcus aureus (MRS A).

36. A method comprising administering an effective amount of the composition of claim 20 to a patient.

37. The method of claim 36, wherein said patient is in need of said composition as a cytokine antagonist; to improve skin rejuvenation; to increase stem-cell mobilization; to increase hematopoiesis; to improve wound healing; to treat, inhibit and/or prevent HIV; to treat, inhibit and/or prevent cancer; to treat, inhibit and/or prevent a bacterial infection; and to treat, inhibit and/or prevent a viral infection.

38. The method of claim 37, wherein said patient is suffering from an HIV infection.

39. The method of claim 38, wherein said HIV is a strain selected from one or more of the group consisting of R5 and X4 from HIV-1.

40. The method of claim 37, wherein said patient is suffering from a bacterial infection.

41. The method of claim 40, wherein said bacterial infection is an infection of Acinetobacter baumannii, Burkholderia Cepacia, Enterococcus faecalis, Escherichia coli, Pseudomonas aeruginosa or Staphylococcus aureus.

42. The method of claim 41, wherein said bacterial infection is an infection of methicillin- resistant Staphylococcus aureus (MRS A).

43. A method of synthesizing a compound of claim 1 or claim 5 comprising addition of a guanide reagent, a biguanide reagent or a phenylguanide reagent to a reactive primary or secondary amine.

44. The method of claim 43, wherein the guanide reagent is O-methylisourea sulfate salt.

45. The method of claim 43, wherein the biguanide reagent is S-methyl- guanylisothiouronium iodide.

46. The method of claim 43, wherein the phenylguanide reagent is S-methyl-N- phenylisothiouronium iodide.

47. The method of claim 43, wherein the compound of claim 1 or claim 5 is linear, branched, or dendrimeric .